Nucleic acids and corresponding proteins entitled 158P3D2 useful in treatment and detection of cancer

ABSTRACT

A novel gene 158P3D2 and its encoded protein, and variants thereof, are described wherein 158P3D2 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 158P3D2 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 158P3D2 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with 158P3D2 can be used in active or passive immunization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of pending U.S. patentapplication Ser. No. 10/107,532, filed 25-Mar.-2002, which claimspriority to U.S. provisional patent application U.S. Ser. No.60/283,112, filed 10-Apr.-2001, and U.S. provisional patent applicationU.S. Ser. No. 60/286,630, filed 25-Apr.-2001. The contents of theapplications listed in this paragraph are fully incorporated byreference herein.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

SUBMISSION ON COMPACT DISC

The content of the following submission on compact discs is incorporatedherein by reference in its entirety: A compact disc copy of the SequenceListing (COPY 1) (file name: 511582006420, date recorded: Nov. 17, 2004,size: 718,848 bytes); and a duplicate compact disc copy of the SequenceListing (COPY 2) (file name: 511582006420, date recorded: Nov. 17, 2004,size: 718,848 bytes).

FIELD OF THE INVENTION

The invention described herein relates to genes and their encodedproteins, termed 158P3D2 and variants thereof, expressed in certaincancers, and to diagnostic and therapeutic methods and compositionsuseful in the management of cancers that express 158P3D2.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease.

Worldwide, millions of people die from cancer every year. In the UnitedStates alone, as reported by the American Cancer Society, cancer causesthe death of well over a half-million people annually, with over 1.2million new cases diagnosed per year. While deaths from heart diseasehave been declining significantly, those resulting from cancer generallyare on the rise. In the early part of the next century, cancer ispredicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most commoncancer in males and is the second leading cause of cancer death in men.In the United States alone, well over 30,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, surgical castration and chemotherapy continue to be the maintreatment modalities. Unfortunately, these treatments are ineffectivefor many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the diagnosis and management of this disease. Although theserum prostate specific antigen (PSA) assay has been a very useful tool,however its specificity and general utility is widely regarded aslacking in several important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC (LosAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCID) miceand have exhibited the capacity to mimic the transition from androgendependence to androgen independence (Klein et al., 1997, Nat. Med.3:402). More recently identified prostate cancer markers include PCTA-1(Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252),prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res1996 Sep. 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad SciUSA. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA)(Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adultmalignancies. Once adenomas reach a diameter of 2 to 3 cm, malignantpotential exists. In the adult, the two principal malignant renal tumorsare renal cell adenocarcinoma and transitional cell carcinoma of therenal pelvis or urethras. The incidence of renal cell adenocarcinoma isestimated at more than 29,000 cases in the United States, and more than11,600 patients died of this disease in 1998. Transitional cellcarcinoma is less frequent, with an incidence of approximately 500 casesper year in the United States.

Surgery has been the primary therapy for renal cell adenocarcinoma formany decades. Until recently, metastatic disease has been refractory toany systemic therapy. With recent developments in systemic therapies,particularly immunotherapies, metastatic renal cell carcinoma may beapproached aggressively in appropriate patients with a possibility ofdurable responses. Nevertheless, there is a remaining need for effectivetherapies for these patients.

Of all new cases of cancer in the United States, bladder cancerrepresents approximately 5 percent in men (fifth most common neoplasm)and 3 percent in women (eighth most common neoplasm). The incidence isincreasing slowly, concurrent with an increasing older population. In1998, there was an estimated 54,500 cases, including 39,500 in men and15,000 in women. The age-adjusted incidence in the United States is 32per 100,000 for men and eight per 100,000 in women. The historicmale/female ratio of 3:1 may be decreasing related to smoking patternsin women. There were an estimated 11,000 deaths from bladder cancer in1998 (7,800 in men and 3,900 in women). Bladder cancer incidence andmortality strongly increase with age and will be an increasing problemas the population becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is managedwith a combination of transurethral resection of the bladder (TUR) andintravesical chemotherapy or immunotherapy. The multifocal and recurrentnature of bladder cancer points out the limitations of TUR. Mostmuscle-invasive cancers are not cured by TUR alone. Radical cystectomyand urinary diversion is the most effective means to eliminate thecancer but carry an undeniable impact on urinary and sexual function.There continues to be a significant need for treatment modalities thatare beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in theUnited States, including 93,800 cases of colon cancer and 36,400 ofrectal cancer. Colorectal cancers are the third most common cancers inmen and women. Incidence rates declined significantly during 1992-1996(−2.1% per year). Research suggests that these declines have been due toincreased screening and polyp removal, preventing progression of polypsto invasive cancers. There were an estimated 56,300 deaths (47,700 fromcolon cancer, 8,600 from rectal cancer) in 2000, accounting for about11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectalcancer, and for cancers that have not spread, it is frequently curative.Chemotherapy, or chemotherapy plus radiation, is given before or aftersurgery to most patients whose cancer has deeply perforated the bowelwall or has spread to the lymph nodes. A permanent colostomy (creationof an abdominal opening for elimination of body wastes) is occasionallyneeded for colon cancer and is infrequently required for rectal cancer.There continues to be a need for effective diagnostic and treatmentmodalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancerin 2000, accounting for 14% of all U.S. cancer diagnoses. The incidencerate of lung and bronchial cancer is declining significantly in men,from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s,the rate of increase among women began to slow. In 1996, the incidencerate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000,accounting for 28% of all cancer deaths. During 1992-1996, mortalityfrom lung cancer declined significantly among men (−1.7% per year) whilerates for women were still significantly increasing (0.9% per year).Since 1987, more women have died each year of lung cancer than breastcancer, which, for over 40 years, was the major cause of cancer death inwomen. Decreasing lung cancer incidence and mortality rates most likelyresulted from decreased smoking rates over the previous 30 years;however, decreasing smoking patterns among women lag behind those ofmen. Of concern, although the declines in adult tobacco use have slowed,tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by thetype and stage of the cancer and include surgery, radiation therapy, andchemotherapy. For many localized cancers, surgery is usually thetreatment of choice. Because the disease has usually spread by the timeit is discovered, radiation therapy and chemotherapy are often needed incombination with surgery. Chemotherapy alone or combined with radiationis the treatment of choice for small cell lung cancer; on this regimen,a large percentage of patients experience remission, which in some casesis long lasting. There is however, an ongoing need for effectivetreatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were expectedto occur among women in the United States during 2000. Additionally,about 1,400 new cases of breast cancer were expected to be diagnosed inmen in 2000. After increasing about 4% per year in the 1980s, breastcancer incidence rates in women have leveled off in the 1990s to about110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women,400 men) in 2000 due to breast cancer. Breast cancer ranks second amongcancer deaths in women. According to the most recent data, mortalityrates declined significantly during 1992-1996 with the largest decreasesin younger women, both white and black. These decreases were probablythe result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient'spreferences, treatment of breast cancer may involve lumpectomy (localremoval of the tumor) and removal of the lymph nodes under the arm;mastectomy (surgical removal of the breast) and removal of the lymphnodes under the arm; radiation therapy; chemotherapy; or hormonetherapy. Often, two or more methods are used in combination. Numerousstudies have shown that, for early stage disease, long-term survivalrates after lumpectomy plus radiotherapy are similar to survival ratesafter modified radical mastectomy. Significant advances inreconstruction techniques provide several options for breastreconstruction after mastectomy. Recently, such reconstruction has beendone at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amountsof surrounding normal breast tissue may prevent the local recurrence ofthe DCIS. Radiation to the breast and/or tamoxifen may reduce the chanceof DCIS occurring in the remaining breast tissue. This is importantbecause DCIS, if left untreated, may develop into invasive breastcancer. Nevertheless, there are serious side effects or sequelae tothese treatments. There is, therefore, a need for efficacious breastcancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the UnitedStates in 2000. It accounts for 4% of all cancers among women and rankssecond among gynecologic cancers. During 1992-1996, ovarian cancerincidence rates were significantly declining. Consequent to ovariancancer, there were an estimated 14,000 deaths in 2000. Ovarian cancercauses more deaths than any other cancer of the female reproductivesystem.

Surgery, radiation therapy, and chemotherapy are treatment options forovarian cancer. Surgery usually includes the removal of one or bothovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus(hysterectomy). In some very early tumors, only the involved ovary willbe removed, especially in young women who wish to have children. Inadvanced disease, an attempt is made to remove all intra-abdominaldisease to enhance the effect of chemotherapy. There continues to be animportant need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in theUnited States in 2000. Over the past 20 years, rates of pancreaticcancer have declined in men. Rates among women have remainedapproximately constant but may be beginning to decline. Pancreaticcancer caused an estimated 28,200 deaths in 2000 in the United States.Over the past 20 years, there has been a slight but significant decreasein mortality rates among men (about −0.9% per year) while rates haveincreased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options forpancreatic cancer. These treatment options can extend survival and/orrelieve symptoms in many patients but are not likely to produce a curefor most. There is a significant need for additional therapeutic anddiagnostic options for pancreatic cancer.

SUMMARY OF THE INVENTION

The present invention relates to a gene, designated 158P3D2, that hasnow been found to be over-expressed in the cancer(s) listed in Table I.Northern blot expression analysis of 158P3D2 gene expression in normaltissues shows a restricted expression pattern in adult tissues. Thenucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of158P3D2 are provided. The tissue-related profile of 158P3D2 in normaladult tissues, combined with the over-expression observed in the tissueslisted in Table I, shows that 158P3D2 is aberrantly over-expressed in atleast some cancers, and thus serves as a useful diagnostic,prophylactic, prognostic, and/or therapeutic target for cancers of thetissue(s) such as those listed in Table I.

The invention provides polynucleotides corresponding or complementary toall or part of the 158P3D2 genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding158P3D2-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80,85, 90, 95, 100 or more than 100 contiguous amino acids of a158P3D2-related protein, as well as the peptides/proteins themselves;DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides oroligonucleotides complementary or having at least a 90% homology to the158P3D2 genes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides that hybridize to the 158P3D2 genes, mRNAs, or to158P3D2-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding 158P3D2. Recombinant DNA moleculescontaining 158P3D2 polynucleotides, cells transformed or transduced withsuch molecules, and host-vector systems for the expression of 158P3D2gene products are also provided. The invention further providesantibodies that bind to 158P3D2 proteins and polypeptide fragmentsthereof, including polyclonal and monoclonal antibodies, murine andother mammalian antibodies, chimeric antibodies, humanized and fullyhuman antibodies, and antibodies labeled with a detectable marker ortherapeutic agent. In certain embodiments, there is a proviso that theentire nucleic acid sequence of FIG. 2 is not encoded and/or the entireamino acid sequence of FIG. 2 is not prepared. In certain embodiments,the entire nucleic acid sequence of FIG. 2 is encoded and/or the entireamino acid sequence of FIG. 2 is prepared, either of which are inrespective human unit dose forms.

The invention further provides methods for detecting the presence andstatus of 158P3D2 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express 158P3D2.A typical embodiment of this invention provides methods for monitoring158P3D2 gene products in a tissue or hematology sample having orsuspected of having some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeuticcompositions and strategies for treating cancers that express 158P3D2such as cancers of tissues listed in Table I, including therapies aimedat inhibiting the transcription, translation, processing or function of158P3D2 as well as cancer vaccines. In one aspect, the inventionprovides compositions, and methods comprising them, for treating acancer that expresses 158P3D2 in a human subject wherein the compositioncomprises a carrier suitable for human use and a human unit dose of oneor more than one agent that inhibits the production or function of158P3D2. Preferably, the carrier is a uniquely human carrier. In anotheraspect of the invention, the agent is a moiety that is immunoreactivewith 158P3D2 protein. Non-limiting examples of such moieties include,but are not limited to, antibodies (such as single chain, monoclonal,polyclonal, humanized, chimeric, or human antibodies), functionalequivalents thereof (whether naturally occurring or synthetic), andcombinations thereof. The antibodies can be conjugated to a diagnosticor therapeutic moiety. In another aspect, the agent is a small moleculeas defined herein.

In another aspect, the agent comprises one or more than one peptidewhich comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLAclass I molecule in a human to elicit a CTL response to 158P3D2 and/orone or more than one peptide which comprises a helper T lymphocyte (HTL)epitope which binds an HLA class II molecule in a human to elicit an HTLresponse. The peptides of the invention may be on the same or on one ormore separate polypeptide molecules. In a further aspect of theinvention, the agent comprises one or more than one nucleic acidmolecule that expresses one or more than one of the CTL or HTL responsestimulating peptides as described above. In yet another aspect of theinvention, the one or more than one nucleic acid molecule may express amoiety that is immunologically reactive with 158P3D2 as described above.The one or more than one nucleic acid molecule may also be, or encodes,a molecule that inhibits production of 158P3D2. Non-limiting examples ofsuch molecules include, but are not limited to, those complementary to anucleotide sequence essential for production of 158P3D2 (e.g. antisensesequences or molecules that form a triple helix with a nucleotide doublehelix essential for 158P3D2 production) or a ribozyme effective to lyse158P3D2 mRNA.

Note that to determine the starting position of any peptide set forth inTables VIII-XXI and XXII to XLIX (collectively HLA Peptide Tables)respective to its parental protein, e.g., variant 1, variant 2, etc.,reference is made to three factors: the particular variant, the lengthof the peptide in an HLA Peptide Table, and the Search Peptides in TableVII. Generally, a unique Search Peptide is used to obtain HLA peptidesof a particular for a particular variant. The position of each SearchPeptide relative to its respective parent molecule is listed in TableVII. Accordingly, if a Search Peptide begins at position “X”, one mustadd the value “X−1” to each position in Tables VIII-XXI and XXII to XLIXto obtain the actual position of the HLA peptides in their parentalmolecule. For example, if a particular Search Peptide begins at position150 of its parental molecule, one must add 150-1, i.e., 149 to each HLApeptide amino acid position to calculate the position of that amino acidin the parent molecule.

One embodiment of the invention comprises an HLA peptide, that occurs atleast twice m Tables VIII-XXI and XXII to XLIX collectively, or anoligonucleotide that encodes the HLA peptide. Another embodiment of theinvention comprises an HLA peptide that occurs at least once in TablesVIII-XXI and at least once in tables XXII to XLIX, or an oligonucleotidethat encodes the HLA peptide.

Another embodiment of the invention is antibody epitopes, which comprisea peptide regions, or an oligonucleotide encoding the peptide region,that has one two, three, four, or five of the following characteristics:

-   -   i) a peptide region of at least 5 amino acids of a particular        peptide of FIG. 3, in any whole number increment up to the full        length of that protein in FIG. 3, that includes an amino acid        position having a value equal to or greater than 0.5, 0.6, 0.7,        0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity        profile of FIG. 5;    -   ii) a peptide region of at least 5 amino acids of a particular        peptide of FIG. 3, in any whole number increment up to the full        length of that protein in FIG. 3, that includes an amino acid        position having a value equal to or less than 0.5, 0.4, 0.3,        0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity        profile of FIG. 6;    -   iii) a peptide region of at least 5 amino acids of a particular        peptide of FIG. 3, in any whole number increment up to the full        length of that protein in FIG. 3, that includes an amino acid        position having a value equal to or greater than 0.5, 0.6, 0.7,        0.8, 0.9, or having a value equal to 1.0, in the Percent        Accessible Residues profile of FIG. 7;    -   iv) a peptide region of at least 5 amino acids of a particular        peptide of FIG. 3, in any whole number increment up to the full        length of that protein in FIG. 3, that includes an amino acid        position having a value equal to or greater than 0.5, 0.6, 0.7,        0.8, 0.9, or having a value equal to 1.0, in the Average        Flexibility profile of FIG. 8; or    -   v) a peptide region of at least 5 amino acids of a particular        peptide of FIG. 3, in any whole number increment up to the full        length of that protein in FIG. 3, that includes an amino acid        position having a value equal to or greater than 0.5, 0.6, 0.7,        0.8, 0.9, or having a value equal to 1.0, in the Beta-turn        profile of FIG. 9[!!! The Figure descriptions need to be revised        when using this as a template].

BRIEF DESCRIPTION OF THE FIGURES FIG. 1. The 158P3D2 SSH sequence of 312nucleotides.

FIG. 2A) The cDNA and amino acid sequence of 158P3D2 variant 1 clone158P3D2-BCP-1 (also called “158P3D2 v.1” or “158P3D2 variant 1”) isshown in FIG. 2A. The start methionine is underlined. The open readingframe extends from nucleic acid 849-1835 including the stop codon.

FIG. 2B) The cDNA and amino acid sequence of 158P3D2 variant 2A (alsocalled “158P3D2 v.2”) is shown in FIG. 2B. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 117-827 including the stop codon.

FIG. 2C) The cDNA and amino acid sequence of 158P3D2 variant 2B (alsocalled “158P3D2 v.2”) is shown in FIG. 2C. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 2249-2794 including the stop codon.

FIG. 2D) The cDNA and amino acid sequence of 158P3D2 variant 3 (alsocalled “158P3D2 v.3”) is shown in FIG. 2D. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 849-1835 including the stop codon.

FIG. 2E) The cDNA and amino acid sequence of 158P3D2 variant 4 (alsocalled “158P3D2 v.4”) is shown in FIG. 2E. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 849-1835 including the stop codon.

FIG. 2F) The cDNA and amino acid sequence of 158P3D2 variant 5A (alsocalled “158P3D2 v.5”) is shown in FIG. 2F. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 849-1385 including the stop codon.

FIG. 2G) The cDNA and amino acid sequence of 158P3D2 variant 5B (alsocalled “158P3D2 v.5”) is shown in FIG. 2G. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 1289-1834 including the stop codon.

FIG. 2H) The cDNA and amino acid sequence of 158P3D2 variant 6 (alsocalled “158P3D2 v.6”) is shown in FIG. 2H. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 849-1835 including the stop codon.

FIG. 21) The cDNA and amino acid sequence of 158P3D2 variant 7 (alsocalled “158P3D2 v.7”) is shown in FIG. 21. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 849-1835 including the stop codon.

FIG. 2J) The cDNA and amino acid sequence of 158P3D2 variant 8 (alsocalled “158P3D2 v.8”) is shown in FIG. 2J. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 849-1835 including the stop codon.

FIG. 2K) The cDNA and amino acid sequence of 158P3D2 variant 14 (alsocalled “158P3D2 v.14”) is shown in FIG. 2K. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 65-4246 including the stop codon.

FIG. 2L) The cDNA and amino acid sequence of 158P3D2 variant 15 (alsocalled “158P3D2 v.15”) is shown in FIG. 2L. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 65-3502 including the stop codon.

FIG. 2M) The cDNA and amino acid sequence of 158P3D2 variant 16 (alsocalled “158P3D2 v.16”) is shown in FIG. 2M. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 65-6037 including the stop codon.

FIG. 2N) The cDNA and amino acid sequence of 158P3D2 variant 17 (alsocalled “158P3D2 v.17”) is shown in FIG. 2N. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 65-6175 including the stop codon.

FIG. 2O) The cDNA and amino acid sequence of 158P3D2 variant 18 (alsocalled “158P3D2 v.18”) is shown in FIG. 20. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 2932-4764 including the stop codon.

FIG. 2P) The cDNA and amino acid sequence of 158P3D2 variant 19 (alsocalled “158P3D2 v.19”) is shown in FIG. 2P. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 65-6001 including the stop codon.

FIG. 2Q) The cDNA and amino acid sequence of 158P3D2 variant 20 (alsocalled “158P3D2 v.20”) is shown in FIG. 2Q. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 65-6121 including the stop codon.

FIG. 2R) 158P3D2 v.9 through v.13, SNP variants of 158P3D2 v.1. The158P3D2 v.9 through v.13 proteins have 1072 amino acids. Variants158P3D2 v.4 through v.20 are variants with single nucleotide differencefrom 158P3D2 v.1. 158P3D2 v.10, v.12 and v.13 proteins differ from158P3D2 v.1 by one amino acid. 158P3D2 v.9 and v.11 proteins code forthe same protein as v.1. Though these SNP variants are shown separately,they can also occur in any combinations and in any of the transcriptvariants listed above in FIGS. 2A-2Q.

FIG. 3A) The amino acid sequence of 158P3D2 v.1 clone 158P3D2-BCP-1 isshown in FIG. 3A; it has 328 amino acids.

FIG. 3B) The amino acid sequence of 158P3D2 v.2A is shown in FIG. 3B; ithas 236 amino acids.

FIG. 3C) The amino acid sequence of 158P3D2 v.2B is shown in FIG. 3C; ithas 181 amino acids.

FIG. 3D) The amino acid sequence of 158P3D2 v.3 is shown in FIG. 3D; ithas 328 amino acids.

FIG. 3E) The amino acid sequence of 158P3D2 v.4 is shown in FIG. 3E; ithas 328 amino acids.

FIG. 3F) The amino acid sequence of 158P3D2 v.5A is shown in FIG. 3F; ithas 178 amino acids.

FIG. 3G) The amino acid sequence of 158P3D2 v.5B is shown in FIG. 3G; ithas 181 amino acids.

FIG. 3H) The amino acid sequence of 158P3D2 v.10 is shown in FIG. 3H; ithas 328 amino acids.

FIG. 3I) The amino acid sequence of 158P3D2 v.11 is shown in FIG. 31; ithas 328 amino acids.

FIG. 3J) The amino acid sequence of 158P3D2 v.12 is shown in FIG. 3J; ithas 328 amino acids.

FIG. 3K) The amino acid sequence of 158P3D2 v.13 is shown in FIG. 3K; ithas 328 amino acids.

FIG. 3L) The amino acid sequence of 158P3D2 v.14 is shown in FIG. 3L; ithas 1393 amino acids.

FIG. 3M) The amino acid sequence of 158P3D2 v.15 is shown in FIG. 3M; ithas 1145 amino acids.

FIG. 3N) The amino acid sequence of 158P3D2 v.16 is shown in FIG. 3N; ithas 1990 amino acids.

FIG. 3O) The amino acid sequence of 158P3D2 v.17 is shown in FIG. 30; ithas 2036 amino acids.

FIG. 3P) The amino acid sequence of 158P3D2 v.18 is shown in FIG. 3P; ithas 610 amino acids.

FIG. 3Q) The amino acid sequence of 158P3D2 v.19 is shown in FIG. 3Q; ithas 1978 amino acids.

FIG. 3R) The amino acid sequence of 158P3D2 v.20 is shown in FIG. 3R; ithas 2018 amino acids.

As used herein, a reference to 158P3D2 includes all variants thereof,including those shown in FIGS. 2, 3, 10, 11, and 12 unless the contextclearly indicates otherwise.

FIG. 4. Omitted.

FIG. 5(a)-(i). Hydrophilicity amino acid profile of 158P3D2 v.1, v.2a,v.2b, v.5a, v.14, v.15, v.16, v.17, and v.18 determined by computeralgorithm sequence analysis using the method of Hopp and Woods (Hopp T.P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828)accessed on the Protscale website located on the World Wide Web at(expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biologyserver.

FIG. 6(a)-(i). Hydropathicity amino acid profile of 158P3D2 v.1, v.2a,v.2b, v.5a, v.14, v.15, v.16, v.17, and v.18 determined by computeralgorithm sequence analysis using the method of Kyte and Doolittle (KyteJ., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132) accessed on theProtScale website located on the World Wide Web at(.expasy.chlcgi-bin/protscale.pl) through the ExPasy molecular biologyserver.

FIG. 7(a)-(i). Percent accessible residues amino acid profile of 158P3D2v.1, v.2a, v.2b, v.5a, v.14, v.15, v.16, v.17, and v.18 determined bycomputer algorithm sequence analysis using the method of Janin (JaninJ., 1979 Nature 277:491-492) accessed on the ProtScale website locatedon the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through theExPasy molecular biology server.

FIG. 8(a)-(i). Average flexibility amino acid profile of 158P3D2 v.1,v.2a, v.2b, v.5a, v.14, v.15, v.16, v.17, and v.18 determined bycomputer algorithm sequence analysis using the method of Bhaskaran andPonnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept.Protein Res. 32:242-255) accessed on the ProtScale website located onthe World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through theExPasy molecular biology server.

FIG. 9(a)-(i). Beta-turn amino acid profile of 158P3D2 v.1, v.2a, v.2b,v.5a, v.14, v.15, v.16, v.17, and v.18 determined by computer algorithmsequence analysis using the method of Deleage and Roux (Deleage, G.,Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScalewebsite located on the World Wide Web at(.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biologyserver.

FIG. 10. Exon compositions of transcript variants of 158P3D2. Variant158P3D2 v.2, v.14 through v.20 are transcript variants. Compared with158P3D2 v.1; v.2 had six additional exons to the 5′ end, an exon 7longer than exon 1 of 158P3D2 v.1 and an exon 10 shorter than exon 4 of158P3D2 v.1. Exons 2, 3, 5, 6 and 7 of 158P3D2 v.1 are the same as exons8, 9, 11, 12 and 13 of 158P3D2 v.2, respectively. Other variants haddifferent exon compositions as shown above. Numbers in “( )” underneaththe box correspond to those of 158P3D2 v.1. Black boxes show the samesequence as 158P3D2 v.1. Length of introns are not proportional.

FIG. 11. Schematic display of protein variants of 158P3D2. Nucleotidevariant 158P3D2 v.2 and 158P3D2 v.5 potentially coded for two differentproteins, designated as variants 158P3D2 v.2A and 158P3D2 v.2B, 158P3D2v.5A and 158P3D2 v.5B, respectively. Variant 158P3D2 v.5B shares thesame amino acid sequence as variant 158P3D2 v.2B. Variants 158P3D2 v.3and v.4 were variants with single amino acid variations. Black box showsthe same sequence as 158P3D2 v.1. Numbers in “( )” underneath the blackboxes correspond to those of 158P3D2 v.1 and those underneath the“brick” boxes correspond to those of v.17. Single amino acid differencesare indicated above the box.

FIG. 12. Schematic display of SNP variants of 158P3D2. Variant 158P3D2v.3 through v.13 are variants with a single nucleotide difference fromv.1. Though these alternative SNP alleles were shown separately, theycould occur in any transcript variants in any combination (calledhaplotype). Numbers in “( )” underneath the box correspond to those of158P3D2 v.1. ‘-’ indicate single nucleotide deletion. Black boxes showthe same sequence as 158P3D2 v.1. SNPs are indicated above the box.

FIG. 13. Secondary structure and transmembrane domains prediction for158P3D2 protein variants.

FIG. 13A (SEQ ID NO: 54), FIG. 13B (SEQ ID NO: 55), FIG. 13C (SEQ ID NO:56), FIG. 13D (SEQ ID NO: 57), FIG. 13E (SEQ ID NO: 58), FIG. 13F (SEQID NO: 59), FIG. 13G (SEQ ID NO: 60), FIG. 13H (SEQ ID NO: 61), FIG. 131(SEQ ID NO: 62): The secondary structures of 158P3D2 protein variants 1,2a, 2b, 5a, 14, 15, 16, 17, 18 respectively, were predicted using theHNN—Hierarchical Neural Network method (NPS@: Network Protein SequenceAnalysis TIBS 2000 March Vol. 25, No 3 [291]:147-150 Combet C., BlanchetC., Geourjon C. and Deleage G., accessed from the ExPasy molecularbiology server. This method predicts the presence and location of alphahelices, extended strands, and random coils from the primary proteinsequence. The percent of the protein variant in a given secondarystructure is also listed.

FIG. 13J, FIG. 13L, FIG. 13N, FIG. 13P, FIG. 13R, FIG. 13T, FIG. 13V,FIG. 13X, and FIG. 13Z: Schematic representation of the probability ofexistence of transmembrane regions of 158P3D2 protein variants 1, 2a,2b, 5a, 14, 15, 16, 17, 18 respectively, based on the TMpred algorithmof Hofmann and Stoffel which utilizes TMBASE (K. Hofmann, W. Stoffel.TMBASE—A database of membrane spanning protein segments Biol. Chem.Hoppe-Seyler 374:166, 1993). FIG. 13K, FIG. 13M, FIG. 130, FIG. 13Q,FIG. 13S, FIG. 13U, FIG. 13W, FIG. 13Y, FIG. 13AA: Schematicrepresentation of the probability of the existence of transmembraneregions of 158P3D2 variants 1, 2a, 2b, 5a, 14, 15, 16, 17, 18respectively, based on the TMHMM algorithm of Sonnhammer, von Heijne,and Krogh (Erik L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: Ahidden Markov model for predicting transmembrane helices in proteinsequences. In Proc. of Sixth Int. Conf. on Intelligent Systems forMolecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R.Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press,1998). The TMpred and TMHMM algorithms are accessed from the ExPasymolecular biology server.

FIG. 14. 158P3D2 Expression in Normal and Cancer Tissue Specimens. Firststrand cDNA was prepared from a panel of 13 normal tissues (brain,heart, kidney, liver, lung, spleen, skeletal muscle, testis, pancreas,colon, stomach) and pools of 4-7 patients from the following cancerindications: bladder, kidney, colon, lung, pancreas, stomach, ovary,breast, multiple cancer metastasis, cervix, lymphoma as well as from apool of patient-derived xenografts (prostate cancer, bladder cancer andkidney cancer). Normalization was performed by PCR using primers toactin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, wasperformed at 26 and 30 cycles of amplification. Samples were run on anagarose gel, and PCR products were quantitated using the AlphaImagersoftware. Results show strong expression of 158P3D2 in cancers of thebladder, kidney, colon, lung, pancreas, stomach, ovary, breast, cervix,and lymphoma. Low expression was detected in all normal tissues testedexcept in normal stomach. Strong expression was also observed in thecancer metastasis pool.

FIG. 15. 158P3D2 Expression in bladder cancer patient specimens. Firststrand cDNA was prepared from normal bladder, bladder cancer cell lines(UM-UC-3, TCCSUP, J82) and a panel of bladder cancer patient specimens.Normalization was performed by PCR using primers to actin and GAPDH.Expression level was recorded as no expression (no signal detected), low(signal detected at 30×), medium (signal detected at 26×), high (strongsignal at 26×). Results show expression of 158P3D2 in the majority ofbladder cancer patient specimens tested. Very low expression wasdetected in normal tissues, but no expression was seen in the cell linestested.

FIG. 16. 158P3D2 Expression in bladder cancer patient specimens bynorthern blotting. RNA was extracted from normal bladder, bladder cancercell lines (UM-UC-3, J82, SCaBER), bladder cancer patient tumors (T) andtheir normal adjacent tissues (NAT). Northern blot with 10 ug of totalRNA were probed with the 158P3D2 sequence. Size standards in kilobasesare on the side. Results show strong expression of 158P3D2 in tumortissues, but not in normal nor NAT tissues.

FIG. 17. 158P3D2 Expression in lung cancer patient specimens. Firststrand cDNA was prepared from normal lung, cancer cell line A427 and apanel of lung cancer patient specimens. Normalization was performed byPCR using primers to actin and GAPDH. Semi-quantitative PCR, usingprimers to 158P3D2, was performed at 26 and 30 cycles of amplification.Expression level was recorded as no expression (no signal detected), low(signal detected at 30×), medium (signal detected at 26×), high (strongsignal at 26×). 158P3D2 is expressed at varying levels in 35/39 (90%) oflung cancer specimens, but not in all 3 normal lung tissues tested.

FIG. 18. 158P3D2 Expression in lung cancer patient specimens by northernblotting. RNA was extracted from normal lung, A427 lung cancer cellline, and a panel of lung cancer patient specimens. Northern blot with10 ug of total RNA were probed with the 158P3D2 sequence. Size standardsin kilobases are on the side. Results show strong expression of 158P3D2in tumor specimens but not in normal tissues.

FIG. 19. 158P3D2 Expression in cancer metastasis patient specimens.First strand cDNA was prepared from normal colon, kidney, liver, lung,pancreas, stomach and from a panel of cancer metastasis patientspecimens. Normalization was performed by PCR using primers to actin andGAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at26 and 30 cycles of amplification. Expression level was recorded as noexpression (no signal detected), low (signal detected at 30×), medium(signal detected at 26×), high (strong signal at 26×). Results showexpression of 158P3D2 in the majority of patient cancer metastasisspecimens tested but not in normal tissues.

FIG. 20. 158P3D2 Expression in cervical cancer patient specimens. Firststrand cDNA was prepared from normal cervix, cervical cancer cell lineHeLa, and a panel of cervical cancer patient specimens. Normalizationwas performed by PCR using primers to actin and GAPDH. Expression levelwas recorded as no expression (no signal detected), low (signal detectedat 30×), medium (signal detected at 26×), high (strong signal at 26×).Results show expression of 158P3D2 in all 14 cervical cancer patientspecimens tested. No expression was detected in normal cervix nor in thecell line tested.

FIG. 21. 158P3D2 Expression in cervical cancer patient specimens bynorthern blotting. RNA was extracted from normal cervix, cervical cancercell line HeLa, and a panel of cervical cancer patient specimens.Northern blot with 10 ug of total RNA were probed with the 158P3D2sequence. Size standards in kilobases are on the side. Results showstrong expression of 158P3D2 in tumor tissues, but not in normal cervixnor in the cell line.

FIG. 22. 158P3D2 Expression in kidney cancer patient specimens. Firststrand cDNA was prepared from normal kidney, kidney cancer cell lines(769-P, A-498, CAKI-1), and a panel of kidney cancer patient specimens.Normalization was performed by PCR using primers to actin and GAPDH.Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and30 cycles of amplification. Expression level was recorded as noexpression (no signal detected), low (signal detected at 30×), medium(signal detected at 26×), high (strong signal at 26×). 158P3D2 isexpressed at varying levels in the majority of kidney cancer patientspecimens, but not in all 3 normal kidney tissues tested. Low expressionwas detected in 2 of 3 cell lines tested.

FIG. 23. 158P3D2 Expression in kidney cancer patient specimens bynorthern blotting. RNA was extracted from normal kidney and a panel ofkidney cancer patient specimens. Northern blot with 10 ug of total RNAwere probed with the 158P3D2 sequence. Size standards in kilobases areon the side. Results show strong expression of 158P3D2 in tumorspecimens but not in the normal tissue.

FIG. 24. 158P3D2 Expression in stomach cancer patient specimens. Firststrand cDNA was prepared from normal stomach, and a panel of stomachcancer patient specimens. Normalization was performed by PCR usingprimers to actin and GAPDH. Semi-quantitative PCR, using primers to158P3D2, was performed at 26 and 30 cycles of amplification. Expressionlevel was recorded as no expression (no signal detected), low (signaldetected at 30×), medium (signal detected at 26×), high (strong signalat 26×). 158P3D2 is expressed at varying levels in the majority ofstomach cancer patient specimens. Weak expression was detected in the 2normal stomach, and only in 1 of the 2 NAT tissues tested.

FIG. 25. 158P3D2 Expression in stomach cancer patient specimens bynorthern blotting. RNA was extracted from normal stomach and a panel ofstomach cancer patient specimens. Northern blot with 10 ug of total RNAwere probed with the 158P3D2 sequence.

Size standards in kilobases are on the side. Results show strongexpression of 158P3D2 in tumor specimens but not in the normal tissue.

FIG. 26. 158P3D2 Expression in colon cancer patient specimens. Firststrand cDNA was prepared from normal colon, colon cancer cell lines(LoVo, CaCO-2, SK CO 1, Colo 205, T284), and a panel of colon cancerpatient specimens. Normalization was performed by PCR using primers toactin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, wasperformed at 26 and 30 cycles of amplification. Expression level wasrecorded as no expression (no signal detected), low (signal detected at30×), medium (signal detected at 26×), high (strong signal at 26×).158P3D2 is expressed at varying levels in the majority of colon cancerpatient specimens. But it was weakly expressed in just 2 of 3 normaltissues, and 3 of 5 cell lines tested.

FIG. 27. 158P3D2 Expression in uterus cancer patient specimens. Firststrand cDNA was prepared from normal uterus and a panel of uterus cancerpatient specimens. Normalization was performed by PCR using primers toactin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, wasperformed at 26 and 30 cycles of amplification. Expression level wasrecorded as no expression (no signal detected), low (signal detected at30×), medium (signal detected at 26×), high (strong signal at 26×).Results show 158P3D2 is expressed at varying levels in the majority ofuterus cancer patient specimens, but not in normal uterus.

FIG. 28. 158P3D2 Expression in breast cancer patient specimens. Firststrand cDNA was prepared from normal breast, breast cancer cell lines(MD-MBA-435S, DU4475, MCF-7, CAMA-1, MCF10A), and a panel of breastcancer patient specimens. Normalization was performed by PCR usingprimers to actin and GAPDH. Semi-quantitative PCR, using primers to158P3D2, was performed at 26 and 30 cycles of amplification. Expressionlevel was recorded as no expression (no signal detected), low (signaldetected at 30×), medium (signal detected at 26×), high (strong signalat 26×). Results show 158P3D2 is expressed at varying levels in themajority of breast cancer patient specimens. But it was weakly expressedin just 2 of 3 normal tissues, and 2 of 5 cell lines tested.

FIG. 29. Serum titer of mice immunized with KLH-peptide encoding aminoacids 315-328 of 158P3D2. Serial dilutions of serum taken from immunizedmice were incubated on an ELISA plate coated with the 158P3 D2 peptideconjugated to ovalbumin. Specific bound antibody was then detected byincubation goat anti-mouse IgG-HRP conjugate and then visualized andquantitated by development with TMB substrate and optical densitydetermination.

FIG. 30. Validation of 158P3D2 siRNA oligo. Cos-1 cells were transfectedwith 1 μg pcDNA3-158P3D2, which encodes a full-length 158P3D2 proteinfusion with a Myc/His tag on the C-terminus, simultaneously withLipofectamine 2000 reagent (LF2k) alone, or with control CT1 oligo (20nM), 158P3D2.b oligo (20 nM), or no DNA or oligo (No DNA). After 72hours, the cells were lysed in 1% Triton buffer, and 50 μg of totalsoluble cell lysate was analyzed by Western blotting. The upper panelwas blotted with anti-Myc (1:1000) to detect the 158P3D2-Myc/His fusionprotein and the lower panel was developed with anti-actin. The level of158P3D2 was diminished by the 158P3D2 siRNA oligo, whereas no change wasobserved with the control (LF2k) or siRNA oligo CT1. In contrast, nochange in the level of actin was noted in the cell lysates, indicatingthat the loading was equivalent in all lanes.

FIG. 31. Effect of 158P3D2 RNAi on cell proliferation. SCaBER cells orCos-1 cells were transfected with Lipofectamine 2000 reagent (LF2K)alone, or with negative control Luc4 oligo (20 nM), positive control Eg5oligo (20 nM) or 158P3D2.b oligo (20 nM). After 48 hours, the media wasreplaced and the cells were incubated for 24 hrs, pulsed with³H-thymidine at 1.5 μCi/ml for 14 hrs, harvested onto a filtermat andcounted in scintillation cocktail on a Microbeta trilux counter.Percentage cell proliferation relative to the LF2k control (100%) isshown. The reduction in 158P3D2 levels by the 158P3D2.b siRNA oligocorrelated with diminished cell proliferation in the SCaBER cells, butno effect was observed in the 158P3D2-negative cell line Cos-1.

DETAILED DESCRIPTION OF THE INVENTION

Outline of Sections

-   -   I.) Definitions    -   II.) 158P3D2 Polynucleotides        -   II.A.) Uses of 158P3D2 Polynucleotides            -   II.A.1.) Monitoring of Genetic Abnormalities            -   II.A.2.) Antisense Embodiments            -   II.A.3.) Primers and Primer Pairs            -   II.A.4.) Isolation of 158P3D2-Encoding Nucleic Acid                Molecules            -   II.A.5.) Recombinant Nucleic Acid Molecules and                Host-Vector Systems    -   III.) 158P3D2-related Proteins        -   III.A.) Motif-bearing Protein Embodiments        -   III.B.) Expression of 158P3D2-related Proteins        -   III.C.) Modifications of 158P3D2-related Proteins        -   III.D.) Uses of 158P3D2-related Proteins    -   IV.) 158P3D2 Antibodies    -   V.) 158P3D2 Cellular Immune Responses    -   VI.) 158P3D2 Transgenic Animals    -   VII.) Methods for the Detection of 158P3D2    -   VIII.) Methods for Monitoring the Status of 158P3D2-related        Genes and Their Products    -   IX.) Identification of Molecules That Interact With 158P3D2    -   X.) Therapeutic Methods and Compositions        -   X.A.) Anti-Cancer Vaccines        -   X.B.) 158P3D2 as a Target for Antibody-Based Therapy        -   X.C.) 158P3D2 as a Target for Cellular Immune Responses            -   X.C.1. Minigene Vaccines            -   X.C.2. Combinations of CTL Peptides with Helper Peptides            -   X.C.3. Combinations of CTL Peptides with T Cell Priming                Agents            -   X.C.4. Vaccine Compositions Comprising DC Pulsed with                CTL and/or HTL Peptides            -   X.D.) Adoptive Immunotherapy            -   X.E.) Administration of Vaccines for Therapeutic or                Prophylactic Purposes    -   XI.) Diagnostic and Prognostic Embodiments of 158P3D2.    -   XII.) Inhibition of 158P3D2 Protein Function        -   XII.A.) Inhibition of 158P3D2 With Intracellular Antibodies        -   XII.B.) Inhibition of 158P3D2 with Recombinant Proteins        -   XII.C.) Inhibition of 158P3D2 Transcription or Translation        -   XII.D.) General Considerations for Therapeutic Strategies    -   XIII.) Identification, Characterization and Use of Modulators of        109P1D1        -   XIII.A.) Methods to Identify and Use Modulators        -   XIII.B.) Gene Expression-related Assays        -   XIII.C.) Expression Monitoring to Identify Compounds that            Modify Gene Expression        -   XIII.D.) Biological Activity-related Assays        -   XIII.E.) High Throughput Screening to Identify Modulators        -   XIII.F.) Use of Soft Agar Growth and Colony Formation to            Identify and Characterize Modulators        -   XIII.G.) Evaluation of Contact Inhibition and Growth Density            Limitation to Identify and Characterize Modulators        -   XIII.H.) Evaluation of Growth Factor or Serum Dependence to            Identify and Characterize Modulators        -   XIII.I.) Use of Tumor-specific Marker Levels to Identify and            Characterize Modulators        -   XIII.J.) Invasiveness into Matrigel to Identify and            Characterize Modulators        -   XIII.K.) Evaluation of Tumor Growth In Vivo to Identify and            Characterize Modulators        -   XIII.L.) In Vitro Assays to Identify and Characterize            Modulators        -   XIII.M.) Binding Assays to Identify and Characterize            Modulators        -   XIII.N.) Competitive Binding to Identify and Characterize            Modulators        -   XIII.O.) Use of Polynucleotides to Down-regulate or Inhibit            a Protein of the Invention.        -   XIII.P.) Inhibitory and Antisense Nucleotides        -   XIII.Q.) Ribozymes        -   XIII.R.) Use of Modulators in Phenotypic Screening        -   XIII.S.) Use of Modulators to Affect Peptides of the            Invention        -   XIII.T.) Methods of Identifying Characterizing            Cancer-associated Sequences    -   XIV.) KITS/Articles of Manufacture        I.) Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

The terms “advanced prostate cancer”, “locally advanced prostatecancer”, “advanced disease” and “locally advanced disease” mean prostatecancers that have extended through the prostate capsule, and are meantto include stage C disease under the American Urological Association(AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, andstage T3-T4 and N+ disease under the TNM (tumor, node, metastasis)system. In general, surgery is not recommended for patients with locallyadvanced disease, and these patients have substantially less favorableoutcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence 158P3D2 (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence 158P3D2. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar orshares similar or corresponding attributes with another molecule (e.g. a158P3D2-related protein). For example, an analog of a 158P3D2 proteincan be specifically bound by an antibody or T cell that specificallybinds to 158P3D2.

The term “antibody” is used in the broadest sense. Therefore, an“antibody” can be naturally occurring or man-made such as monoclonalantibodies produced by conventional hybridoma technology. Anti-158P3D2antibodies comprise monoclonal and polyclonal antibodies as well asfragments containing the antigen-binding domain and/or one or morecomplementarity determining regions of these antibodies.

An “antibody fragment” is defined as at least a portion of the variableregion of the immunoglobulin molecule that binds to its target, i.e.,the antigen-binding region. In one embodiment it specifically coverssingle anti-158P3D2 antibodies and clones thereof (including agonist,antagonist and neutralizing antibodies) and anti-158P3D2 antibodycompositions with polyepitopic specificity.

The term “codon optimized sequences” refers to nucleotide sequences thathave been optimized for a particular host species by replacing anycodons having a usage frequency of less than about 20%. Nucleotidesequences that have been optimized for expression in a given hostspecies by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as an “expression enhancedsequences.”

A “combinatorial library” is a collection of diverse chemical compoundsgenerated by either chemical synthesis or biological synthesis bycombining a number of chemical “building blocks” such as reagents. Forexample, a linear combinatorial chemical library, such as a polypeptide(e.g., mutein) library, is formed by combining a set of chemicalbuilding blocks called amino acids in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptidecompound). Numerous chemical compounds are synthesized through suchcombinatorial mixing of chemical building blocks (Gallop et al., J. Med.Chem. 37(9): 1233-1251 (1994)).

Preparation and screening of combinatorial libraries is well known tothose of skill in the art. Such combinatorial chemical librariesinclude, but are not limited to, peptide libraries (see, e.g., U.S. Pat.No. 5,010,175, Furka, Pept. Prot. Res. 37:487-493 (1991), Houghton etal., Nature, 354:84-88 (1991)), peptoids (PCT Publication No WO91/19735), encoded peptides (PCT Publication WO 93/20242), randombio-oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Pat.No. 5,288,514), diversomers such as hydantoins, benzodiazepines anddipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913(1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc.114:6568 (1992)), nonpeptidal peptidomimetics with a Beta-D-Glucosescaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218(1992)), analogous organic syntheses of small compound libraries (Chenet al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho, etal., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell etal., J. Org. Chem. 59:658 (1994)). See, generally, Gordon et al., J.Med. Chem. 37:1385 (1994), nucleic acid libraries (see, e.g.,Stratagene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Pat.No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., NatureBiotechnology 14(3): 309-314 (1996), and PCT/US96/10287), carbohydratelibraries (see, e.g., Liang et al., Science 274:1520-1522 (1996), andU.S. Pat. No. 5,593,853), and small organic molecule libraries (see,e.g., benzodiazepines, Baum, C&EN, January 18, page 33 (1993);isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones andmetathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, U.S. Pat. No. 5,288,514; and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 NIPS, 390 NIPS, Advanced Chem Tech, LouisvilleKy.; Symphony, Rainin, Woburn, Mass.; 433A, Applied Biosystems, FosterCity, Calif.; 9050, Plus, Millipore, Bedford, NIA). A number ofwell-known robotic systems have also been developed for solution phasechemistries. These systems include automated workstations such as theautomated synthesis apparatus developed by Takeda Chemical Industries,LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms(Zymate H, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard,Palo Alto, Calif.), which mimic the manual synthetic operationsperformed by a chemist. Any of the above devices are suitable for usewith the present invention. The nature and implementation ofmodifications to these devices (if any) so that they can operate asdiscussed herein will be apparent to persons skilled in the relevantart. In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J.; Asinex,Moscow, RU; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd, Moscow, RU; 3DPharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md.; etc.).

The term “cytotoxic agent” refers to a substance that inhibits orprevents the expression activity of cells, function of cells and/orcauses destruction of cells. The term is intended to include radioactiveisotopes chemotherapeutic agents, and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof. Examples ofcytotoxic agents include, but are not limited to auristatins,auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A-chain,combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin,taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracindione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40,abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin,mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin,calicheamicin, Sapaonaria officinalis inhibitor, and glucocorticoid andother chemotherapeutic agents, as well as radioisotopes such as At211,I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32 and radioactiveisotopes of Lu including Lu177. Antibodies may also be conjugated to ananti-cancer pro-drug activating enzyme capable of converting thepro-drug to its active form.

The “gene product” is sometimes referred to herein as a protein or mRNA.For example, a “gene product of the invention” is sometimes referred toherein as a “cancer amino acid sequence”, “cancer protein”, “protein ofa cancer listed in Table I”, a “cancer mRNA”, “mRNA of a cancer listedin Table I”, etc. In one embodiment, the cancer protein is encoded by anucleic acid of FIG. 2. The cancer protein can be a fragment, oralternatively, be the full-length protein to the fragment encoded by thenucleic acids of FIG. 2. In one embodiment, a cancer amino acid sequenceis used to determine sequence identity or similarity. In anotherembodiment, the sequences are naturally occurring allelic variants of aprotein encoded by a nucleic acid of FIG. 2. In another embodiment, thesequences are sequence variants as further described herein.

“High throughput screening” assays for the presence, absence,quantification, or other properties of particular nucleic acids orprotein products are well known to those of skill in the art. Similarly,binding assays and reporter gene assays are similarly well known. Thus,e.g., U.S. Pat. No. 5,559,410 discloses high throughput screeningmethods for proteins; U.S. Pat. No. 5,585,639 discloses high throughputscreening methods for nucleic acid binding (i.e., in arrays); while U.S.Pat. Nos. 5,576,220 and 5,541,061 disclose high throughput methods ofscreening for ligand/antibody binding.

In addition, high throughput screening systems are commerciallyavailable (see, e.g., Amersham Biosciences, Piscataway, N.J.; ZymarkCorp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; BeckmanInstruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick,Mass.; etc.). These systems typically automate entire procedures,including all sample and reagent pipetting, liquid dispensing, timedincubations, and final readings of the microplate in detector(s)appropriate for the assay. These configurable systems provide highthroughput and rapid start up as well as a high degree of flexibilityand customization. The manufacturers of such systems provide detailedprotocols for various high throughput systems. Thus, e.g., Zymark Corp.provides technical bulletins describing screening systems for detectingthe modulation of gene transcription, ligand binding, and the like.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, e.g., Stites, et al.,Immunology, 8th Ed., Lange Publishing, Los Altos, Calif. (1994).

The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used inthe context of polynucleotides, are meant to refer to conventionalhybridization conditions, preferably such as hybridization in 50%formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures forhybridization are above 37 degrees C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55 degrees C.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment. For example, a polynucleotide is said to be “isolated” whenit is substantially separated from contaminant polynucleotides thatcorrespond or are complementary to genes other than the 158P3D2 genes orthat encode polypeptides other than 158P3D2 gene product or fragmentsthereof. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated 158P3D2 polynucleotide. A protein issaid to be “isolated,” for example, when physical, mechanical orchemical methods are employed to remove the 158P3D2 proteins fromcellular constituents that are normally associated with the protein. Askilled artisan can readily employ standard purification methods toobtain an isolated 158P3D2 protein. Alternatively, an isolated proteincan be prepared by chemical means.

The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

The terms “metastatic prostate cancer” and “metastatic disease” meanprostate cancers that have spread to regional lymph nodes or to distantsites, and are meant to include stage D disease under the AUA system andstage T×N×M+ under the TNM system. As is the case with locally advancedprostate cancer, surgery is generally not indicated for patients withmetastatic disease, and hormonal (androgen ablation) therapy is apreferred treatment modality. Patients with metastatic prostate cancereventually develop an androgen-refractory state within 12 to 18 monthsof treatment initiation. Approximately half of these androgen-refractorypatients die within 6 months after developing that status. The mostcommon site for prostate cancer metastasis is bone. Prostate cancer bonemetastases are often osteoblastic rather than osteolytic (i.e.,resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

The term “modulator” or “test compound” or “drug candidate” orgrammatical equivalents as used herein describe any molecule, e.g.,protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, etc., to be tested for the capacity to directly orindirectly alter the cancer phenotype or the expression of a cancersequence, e.g., a nucleic acid or protein sequences, or effects ofcancer sequences (e.g., signaling, gene expression, protein interaction,etc.) In one aspect, a modulator will neutralize the effect of a cancerprotein of the invention. By “neutralize” is meant that an activity of aprotein is inhibited or blocked, along with the consequent effect on thecell. In another aspect, a modulator will neutralize the effect of agene, and its corresponding protein, of the invention by normalizinglevels of said protein. In preferred embodiments, modulators alterexpression profiles, or expression profile nucleic acids or proteinsprovided herein, or downstream effector pathways. In one embodiment, themodulator suppresses a cancer phenotype, e.g. to a normal tissuefingerprint. In another embodiment, a modulator induced a cancerphenotype. Generally, a plurality of assay mixtures is run in parallelwith different agent concentrations to obtain a differential response tothe various concentrations. Typically, one of these concentrationsserves as a negative control, i.e., at zero concentration or below thelevel of detection.

Modulators, drug candidates or test compounds encompass numerouschemical classes, though typically they are organic molecules,preferably small organic compounds having a molecular weight of morethan 100 and less than about 2,500 Daltons. Preferred small moleculesare less than 2000, or less than 1500 or less than 1000 or less than 500D. Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Modulators also comprise biomolecules such aspeptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof. Particularlypreferred are peptides. One class of modulators are peptides, forexample of from about five to about 35 amino acids, with from about fiveto about 20 amino acids being preferred, and from about 7 to about 15being particularly preferred. Preferably, the cancer modulatory proteinis soluble, includes a non-transmembrane region, and/or, has anN-terminal Cys to aid in solubility. In one embodiment, the C-terminusof the fragment is kept as a free acid and the N-terminus is a freeamine to aid in coupling, i.e., to cysteine. In one embodiment, a cancerprotein of the invention is conjugated to an immunogenic agent asdiscussed herein. In one embodiment, the cancer protein is conjugated toBSA. The peptides of the invention, e.g., of preferred lengths, can belinked to each other or to other amino acids to create a longerpeptide/protein. The modulatory peptides can be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. In a preferred embodiment, peptide/protein-basedmodulators are antibodies, and fragments thereof, as defined herein.

Modulators of cancer can also be nucleic acids. Nucleic acid modulatingagents can be naturally occurring nucleic acids, random nucleic acids,or “biased” random nucleic acids. For example, digests of prokaryotic oreukaryotic genomes can be used in an approach analogous to that outlinedabove for proteins.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the antibodiescomprising the population are identical except for possible naturallyoccurring mutations that are present in minor amounts.

A “motif”, as in biological motif of a 158P3D2-related protein, refersto any pattern of amino acids forming part of the primary sequence of aprotein, that is associated with a particular function (e.g.protein-protein interaction, protein-DNA interaction, etc) ormodification (e.g. that is phosphorylated, glycosylated or amidated), orlocalization (e.g. secretory sequence, nuclear localization sequence,etc.) or a sequence that is correlated with being immunogenic, eitherhumorally or cellularly. A motif can be either contiguous or capable ofbeing aligned to certain positions that are generally correlated with acertain function or property. In the context of HLA motifs, “motif”refers to the pattern of residues in a peptide of defined length,usually a peptide of from about 8 to about 13 amino acids for a class IHLA motif and from about 6 to about 25 amino acids for a class II HLAmotif, which is recognized by a particular HLA molecule. Peptide motifsfor HLA binding are typically different for each protein encoded by eachhuman HLA allele and differ in the pattern of the primary and secondaryanchor residues.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/orcomposition that is physiologically compatible with humans or othermammals.

The term “polynucleotide” means a polymeric form of nucleotides of atleast 10 bases or base pairs in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, and ismeant to include single and double stranded forms of DNA and/or RNA. Inthe art, this term if often used interchangeably with “oligonucleotide”.A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T), as shown for example in FIG. 2, can also beuracil (U); this definition pertains to the differences between thechemical structures of DNA and RNA, in particular the observation thatone of the four major bases in RNA is uracil (U) instead of thymidine(T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or8 amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used. In the art, thisterm is often used interchangeably with “peptide” or “protein”.

An HLA “primary anchor residue” is an amino acid at a specific positionalong a peptide sequence which is understood to provide a contact pointbetween the immunogenic peptide and the HLA molecule. One to three,usually two, primary anchor residues within a peptide of defined lengthgenerally defines a “motif” for an immunogenic peptide. These residuesare understood to fit in close contact with peptide binding groove of anHLA molecule, with their side chains buried in specific pockets of thebinding groove. In one embodiment, for example, the primary anchorresidues for an HLA class I molecule are located at position 2 (from theamino terminal position) and at the carboxyl terminal position of a 8,9, 10, 11, or 12 residue peptide epitope in accordance with theinvention. Alternatively, in another embodiment, the primary anchorresidues of a peptide binds an HLA class II molecule are spaced relativeto each other, rather than to the termini of a peptide, where thepeptide is generally of at least 9 amino acids in length. The primaryanchor positions for each motif and supermotif are set forth in TableIV. For example, analog peptides can be created by altering the presenceor absence of particular residues in the primary and/or secondary anchorpositions shown in Table IV. Such analogs are used to modulate thebinding affinity and/or population coverage of a peptide comprising aparticular HLA motif or supermotif.

“Radioisotopes” include, but are not limited to the following(non-limiting exemplary uses are also set forth):

Examples of Medical Isotopes

Isotope Description of use Actinium-225 See Thorium-229 (Th-229)(AC-225) Actinium-227 Parent of Radium-223 (Ra-223) which is an alphaemitter (AC-227) used to treat metastases in the skeleton resulting fromcancer (i.e., breast and prostate cancers), and cancerradioimmunotherapy Bismuth-212 See Thorium-228 (Th-228) (Bi-212)Bismuth-213 See Thorium-229 (Th-229) (Bi-213) Cadmium-109 Cancerdetection (Cd-109) Cobalt-60 Radiation source for radiotherapy ofcancer, for food (Co-60) irradiators, and for sterilization of medicalsupplies Copper-64 A positron emitter used for cancer therapy and SPECT(Cu-64) imaging Copper-67 Beta/gamma emitter used in cancerradioimmunotherapy (Cu-67) and diagnostic studies (i.e., breast andcolon cancers, and lymphoma) Dysprosium- Cancer radioimmunotherapy 166(Dy-166) Erbium-169 Rheumatoid arthritis treatment, particularly for thesmall (Er-169) joints associated with fingers and toes Europium-152Radiation source for food irradiation and for sterilization (Eu-152) ofmedical supplies Europium-154 Radiation source for food irradiation andfor sterilization (Eu-154) of medical supplies Gadolinium- Osteoporosisdetection and nuclear medical quality 153 (Gd-153) assurance devicesGold-198 Implant and intracavity therapy of ovarian, prostate, and(Au-198) brain cancers Holmium-166 Multiple myeloma treatment intargeted skeletal therapy, (Ho-166) cancer radioimmunotherapy, bonemarrow ablation, and rheumatoid arthritis treatment Iodine-125Osteoporosis detection, diagnostic imaging, tracer drugs, (I-125) braincancer treatment, radiolabeling, tumor imaging, mapping of receptors inthe brain, interstitial radiation therapy, brachytherapy for treatmentof prostate cancer, determination of glomerular filtration rate (GFR),determination of plasma volume, detection of deep vein thrombosis of thelegs Iodine-131 Thyroid function evaluation, thyroid disease detection,(I-131) treatment of thyroid cancer as well as other non-malignantthyroid diseases (i.e., Graves disease, goiters, and hyperthyroidism),treatment of leukemia, lymphoma, and other forms of cancer (e.g., breastcancer) using radioimmunotherapy Iridium-192 Brachytherapy, brain andspinal cord tumor treatment, (Ir-192) treatment of blocked arteries(i.e., arteriosclerosis and restenosis), and implants for breast andprostate tumors Lutetium-177 Cancer radioimmunotherapy and treatment ofblocked (Lu-177) arteries (i.e., arteriosclerosis and restenosis)Molybdenum- Parent of Technetium-99m (Tc-99m) which is used for 99(Mo-99) imaging the brain, liver, lungs, heart, and other organs.Currently, Tc-99m is the most widely used radioisotope used fordiagnostic imaging of various cancers and diseases involving the brain,heart, liver, lungs; also used in detection of deep vein thrombosis ofthe legs Osmium-194 Cancer radioimmunotherapy (Os-194) Palladium-103Prostate cancer treatment (Pd-103) Platinum-195m Studies onbiodistribution and metabolism of cisplatin, a (Pt-195m)chemotherapeutic drug Phosphorus-32 Polycythemia rubra vera (blood celldisease) and (P-32) leukemia treatment, bone cancer diagnosis/treatment;colon, pancreatic, and liver cancer treatment; radiolabeling nucleicacids for in vitro research, diagnosis of superficial tumors, treatmentof blocked arteries (i.e., arteriosclerosis and restenosis), andintracavity therapy Phosphorus-33 Leukemia treatment, bone diseasediagnosis/treatment, (P-33) radiolabeling, and treatment of blockedarteries (i.e., arteriosclerosis and restenosis) Radium-223 SeeActinium-227 (Ac-227) (Ra-223) Rhenium-186 Bone cancer pain relief,rheumatoid arthritis treatment, (Re-186) and diagnosis and treatment oflymphoma and bone, breast, colon, and liver cancers usingradioimmunotherapy Rhenium-188 Cancer diagnosis and treatment using(Re-188) radioimmunotherapy, bone cancer pain relief, treatment ofrheumatoid arthritis, and treatment of prostate cancer Rhodium-105Cancer radioimmunotherapy (Rh-105) Samarium-145 Ocular cancer treatment(Sm-145) Samarium-153 Cancer radioimmunotherapy and bone cancer painrelief (Sm-153) Scandium-47 Cancer radioimmunotherapy and bone cancerpain relief (Sc-47) Selenium-75 Radiotracer used in brain studies,imaging of adrenal (Se-75) cortex by gamma-scintigraphy, laterallocations of steroid secreting tumors, pancreatic scanning, detection ofhyperactive parathyroid glands, measure rate of bile acid loss from theendogenous pool Strontium-85 Bone cancer detection and brain scans(Sr-85) Strontium-89 Bone cancer pain relief, multiple myelomatreatment, and (Sr-89) osteoblastic therapy Technetium- SeeMolybdenum-99 (Mo-99) 99m (Tc-99m) Thorium-228 Parent of Bismuth-212(Bi-212) which is an alpha emitter (Th-228) used in cancerradioimmunotherapy Thorium-229 Parent of Actinium-225 (Ac-225) andgrandparent of (Th-229) Bismuth-213 (Bi-213) which are alpha emittersused in cancer radioimmunotherapy Thulium-170 Gamma source for bloodirradiators, energy source for (Tm-170) implanted medical devicesTin-117m Cancer immunotherapy and bone cancer pain relief (Sn-117m)Tungsten-188 Parent for Rhenium-188 (Re-188) which is used for (W-188)cancer diagnostics/treatment, bone cancer pain relief, rheumatoidarthritis treatment, and treatment of blocked arteries (i.e.,arteriosclerosis and restenosis) Xenon-127 Neuroimaging of braindisorders, high resolution SPECT (Xe-127) studies, pulmonary functiontests, and cerebral blood flow studies Ytterbium-175 Cancerradioimmunotherapy (Yb-175) Yttrium-90 Microseeds obtained fromirradiating Yttrium-89 (Y-89) (Y-90) for liver cancer treatmentYttrium-91 A gamma-emitting label for Yttrium-90 (Y-90) which is (Y-91)used for cancer radioimmunotherapy (i.e., lymphoma, breast, colon,kidney, lung, ovarian, prostate, pancreatic, and inoperable livercancers)

By “randomized” or grammatical equivalents as herein applied to nucleicacids and proteins is meant that each nucleic acid and peptide consistsof essentially random nucleotides and amino acids, respectively. Theserandom peptides (or nucleic acids, discussed herein) can incorporate anynucleotide or amino acid at any position. The synthetic process can bedesigned to generate randomized proteins or nucleic acids, to allow theformation of all or most of the possible combinations over the length ofthe sequence, thus forming a library of randomized candidate bioactiveproteinaceous agents.

In one embodiment, a library is “fully randomized,” with no sequencepreferences or constants at any position. In another embodiment, thelibrary is a “biased random” library. That is, some positions within thesequence either are held constant, or are selected from a limited numberof possibilities. For example, the nucleotides or amino acid residuesare randomized within a defined class, e.g., of hydrophobic amino acids,hydrophilic residues, sterically biased (either small or large)residues, towards the creation of nucleic acid binding domains, thecreation of cysteines, for cross-linking, prolines for SH-3 domains,serines, threonines, tyrosines or histidines for phosphorylation sites,etc., or to purines, etc.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that hasbeen subjected to molecular manipulation in vitro.

Non-limiting examples of small molecules include compounds that bind orinteract with 158P3D2, ligands including hormones, neuropeptides,chemokines, odorants, phospholipids, and functional equivalents thereofthat bind and preferably inhibit 158P3D2 protein function. Suchnon-limiting small molecules preferably have a molecular weight of lessthan about 10 kDa, more preferably below about 9, about 8, about 7,about 6, about 5 or about 4 kDa. In certain embodiments, small moleculesphysically associate with, or bind, 158P3D2 protein; are not found innaturally occurring metabolic pathways; and/or are more soluble inaqueous than non-aqueous solutions.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by, but not limited to, those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C. “Moderately stringent conditions” are described by, but not limitedto, those in Sambrook et al., Molecular Cloning: A Laboratory Manual,New York: Cold Spring Harbor Press, 1989, and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS) less stringent than those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

An HLA “supermotif” is a peptide binding specificity shared by HLAmolecules encoded by two or more HLA alleles. Overall phenotypicfrequencies of HLA-supertypes in different ethnic populations are setforth in Table IV (F). The non-limiting constituents of varioussupetypes are as follows:

-   -   A2: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*6802,        A*6901, A*0207    -   A3: A3, A11, A31, A*3301, A*6801, A*0301, A*1101, A*3101    -   B7: B7, B*3501-03, B*51, B*5301, B*5401, B*5501, B*5502, B*5601,        B*6701, B*7801, B*0702, B*5101, B*5602    -   B44: B*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006)    -   A1: A*0102, A*2604, A*3601, A*4301, A*8001    -   A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003    -   B27: B*1401-O₂, B*1503, B*1509, B*1510, B*1518, B*3801-02,        B*3901, B*3902, B*3903-04, B*4801-02, B*7301, B*2701-08    -   B58: B*1516, B*1517, B*5701, B*5702, B58    -   B62: B*4601, B52, B*1501 (B62), B*1502 (B75), B*1513 (B77)

Calculated population coverage afforded by different HLA-supertypecombinations are set forth in Table IV (G).

As used herein “to treat” or “therapeutic” and grammatically relatedterms, refer to any improvement of any consequence of disease, such asprolonged survival, less morbidity, and/or a lessening of side effectswhich are the byproducts of an alternative therapeutic modality; fulleradication of disease is not required.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cellsthat contain a transgene, which transgene was introduced into the animalor an ancestor of the animal at a prenatal, e.g., an embryonic stage. A“transgene” is a DNA that is integrated into the genome of a cell fromwhich a transgenic animal develops.

As used herein, an HLA or cellular immune response “vaccine” is acomposition that contains or encodes one or more peptides of theinvention. There are numerous embodiments of such vaccines, such as acocktail of one or more individual peptides; one or more peptides of theinvention comprised by a polyepitopic peptide; or nucleic acids thatencode such individual peptides or polypeptides, e.g., a minigene thatencodes a polyepitopic peptide. The “one or more peptides” can includeany whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides ofthe invention. The peptides or polypeptides can optionally be modified,such as by lipidation, addition of targeting or other sequences. HLAclass I peptides of the invention can be admixed with, or linked to, HLAclass II peptides, to facilitate activation of both cytotoxic Tlymphocytes and helper T lymphocytes. HLA vaccines can also comprisepeptide-pulsed antigen presenting cells, e.g., dendritic cells.

The term “variant” refers to a molecule that exhibits a variation from adescribed type or norm, such as a protein that has one or more differentamino acid residues in the corresponding position(s) of a specificallydescribed protein (e.g. the 158P3D2 protein shown in FIG. 2 or FIG. 3.An analog is an example of a variant protein. Splice isoforms and singlenucleotides polymorphisms (SNPs) are further examples of variants.

The “158P3D2-related proteins” of the invention include thosespecifically identified herein, as well as allelic variants,conservative substitution variants, analogs and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein or readily available in the art.Fusion proteins that combine parts of different 158P3D2 proteins orfragments thereof, as well as fusion proteins of a 158P3D2 protein and aheterologous polypeptide are also included. Such 158P3D2 proteins arecollectively referred to as the 158P3D2-related proteins, the proteinsof the invention, or 158P3D2. The term “158P3D2-related protein” refersto a polypeptide fragment or a 158P3D2 protein sequence of 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, ormore than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65,70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275,300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 576 ormore amino acids.

II.) 158P3D2 Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a 158P3D2 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a 158P3D2-related protein and fragments thereof, DNA, RNA,DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to a 158P3D2 gene or mRNA sequence or apart thereof, and polynucleotides or oligonucleotides that hybridize toa 158P3D2 gene, mRNA, or to a 158P3D2 encoding polynucleotide(collectively, “158P3D2 polynucleotides”). In all instances whenreferred to in this section, T can also be U in FIG. 2.

Embodiments of a 158P3D2 polynucleotide include: a 158P3D2polynucleotide having the sequence shown in FIG. 2, the nucleotidesequence of 158P3D2 as shown in FIG. 2 wherein T is U; at least 10contiguous nucleotides of a polynucleotide having the sequence as shownin FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotidehaving the sequence as shown in FIG. 2 where T is U. For example,embodiments of 158P3D2 nucleotides comprise, without limitation:

-   -   (I) a polynucleotide comprising, consisting essentially of, or        consisting of a sequence as shown in FIG. 2, wherein T can also        be U;    -   (II) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2A, from nucleotide        residue number 849 through nucleotide residue number 1835,        including the stop codon, wherein T can also be U;    -   (III) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2B, from nucleotide        residue number 117 through nucleotide residue number 827,        including the stop codon, wherein T can also be U;    -   (IV) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2C, from nucleotide        residue number 2249 through nucleotide residue number 2794,        including the a stop codon, wherein T can also be U;    -   (V) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2D, from nucleotide        residue number 849 through nucleotide residue number 1835,        including the stop codon, wherein T can also be U;    -   (VI) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2E, from nucleotide        residue number 849 through nucleotide residue number 1835,        including the stop codon, wherein T can also be U;    -   (VII) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2F, from nucleotide        residue number 849 through nucleotide residue number 1835,        including the stop codon, wherein T can also be U;    -   (VIII) a polynucleotide comprising, consisting essentially of,        or consisting of the sequence as shown in FIG. 2G, from        nucleotide residue number 1289 through nucleotide residue number        1834, including the stop codon, wherein T can also be U;    -   (IX) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2H, from nucleotide        residue number 849 through nucleotide residue number 1835,        including the stop codon, wherein T can also be U;    -   (X) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 21, from nucleotide        residue number 849 through nucleotide residue number 1835,        including the stop codon, wherein T can also be U;    -   (XI) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2J, from nucleotide        residue number 849 through nucleotide residue number 1835,        including the stop codon, wherein T can also be U;    -   (XII) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2K, from nucleotide        residue number 65 through nucleotide residue number 4246,        including the stop codon, wherein T can also be U;    -   (XIII) a polynucleotide comprising, consisting essentially of,        or consisting of the sequence as shown in FIG. 2L, from        nucleotide residue number 65 through nucleotide residue number        3502, including the stop codon, wherein T can also be U;    -   (XIV) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2M, from nucleotide        residue number 65 through nucleotide residue number 6037,        including the stop codon, wherein T can also be U;    -   (XV) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2N, from nucleotide        residue number 65 through nucleotide residue number 6175,        including the stop codon, wherein T can also be U;    -   (XVI) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 20, from nucleotide        residue number 2932 through nucleotide residue number 4764,        including the stop codon, wherein T can also be U;    -   (XVII) a polynucleotide comprising, consisting essentially of,        or consisting of the sequence as shown in FIG. 2P, from        nucleotide residue number 65 through nucleotide residue number        6001, including the stop codon, wherein T can also be U;    -   (XVIII) a polynucleotide comprising, consisting essentially of,        or consisting of the sequence as shown in FIG. 2Q, from        nucleotide residue number 65 through nucleotide residue number        6121, including the stop codon, wherein T can also be U;    -   (XIX) a polynucleotide that encodes a 158P3D2-related protein        that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%        homologous to an entire amino acid sequence shown in FIG. 2A-Q;    -   (XX) a polynucleotide that encodes a 158P3D2-related protein        that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%        identical to an entire amino acid sequence shown in FIG. 2A-Q;    -   (XXI) a polynucleotide that encodes at least one peptide set        forth in Tables VIII-XXI and XXII-XLIX;    -   (XXII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIGS. 3A-3R in any whole number increment        up to 328, 236, 181, 178, 181, 1393, 1145, 1990, 2036, 610,        1978, and 2018 that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,        26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s)        having a value greater than 0.5 in the Hydrophilicity profile of        FIG. 5;    -   (XXIII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIGS. 3A-3R in any whole number increment        up to 328, 236, 181, 178, 181, 1393, 1145, 1990, 2036, 610,        1978, and 2018 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XXIV) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIGS. 3A-3R in any whole number increment        up to 32.8, 236, 181, 178, 181, 1393, 1145, 1990, 2036, 610,        1978, and 2018 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value greater than 0.5 in the Percent Accessible Residues        profile of FIG. 7;    -   (XXV) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3A-3R in any whole number increment        up to 328, 236, 181, 178, 181, 1393, 1145, 1990, 2036, 610,        1978, and 2018 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value greater than 0.5 in the Average Flexibility profile of        FIG. 8;    -   (XXVI) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3A-3R in any whole number increment        up to 328, 236, 181, 178, 181, 1393, 1145, 1990, 2036, 610,        1978, and 2018 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value greater than 0.5 in the Beta-turn profile of FIG. 9;    -   (XXVII) a polynucleotide that is fully complementary to a        polynucleotide of any one of (I)-(XXVI);    -   (XXVIII) a polynucleotide that is fully complementary to a        polynucleotide of any one of (I)-(XXVII);    -   (XXIX) a peptide that is encoded by any of (I) to (XXVIII); and;    -   (XXX) a composition comprising a polynucleotide of any of        (I)-(XXVIII) or peptide of (XXIX) together with a pharmaceutical        excipient and/or in a human unit dose form;    -   (XXXI) a method of using a polynucleotide of any (I)-(XXVIII) or        peptide of (XXIX) or a composition of (XXX) in a method to        modulate a cell expressing 158P3D2;    -   (XXXII) a method of using a polynucleotide of any (I)-(XXVIII)        or peptide of (XXIX) or a composition of (XXX) in a method to        diagnose, prophylax, prognose, or treat an individual who bears        a cell expressing 158P3D2;    -   (XXIII) a method of using a polynucleotide of any (I)-(XXVIII)        or peptide of (XXIX) or a composition of (XXX) in a method to        diagnose, prophylax, prognose, or treat an individual who bears        a cell expressing 158P3D2, said cell from a cancer of a tissue        listed in Table I;    -   (XXXIV) a method of using a polynucleotide of any (I)-(XXVIII)        or peptide of (XXIX) or a composition of (XXX) in a method to        diagnose, prophylax, prognose, or treat a a cancer;    -   (XXXV) a method of using a polynucleotide of any (I)-(XXVIII) or        peptide of (XXIX) or a composition of (XXX) in a method to        diagnose, prophylax, prognose, or treat a a cancer of a tissue        listed in Table I; and;    -   (XXXVI) a method of using a polynucleotide of any (I)-(XXVIII)        or peptide of (XXIX) or a composition of (XXX) in a method to        identify or characterize a modulator of a cell expressing        158P3D2.

As used herein, a range is understood to disclose specifically all wholeunit positions thereof.

Typical embodiments of the invention disclosed herein include 158P3D2polynucleotides that encode specific portions of 158P3D2 mRNA sequences(and those which are complementary to such sequences) such as those thatencode the proteins and/or fragments thereof, for example: [!!! reviseto include up to the appropriate length].

-   -   (a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,        80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,        145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225,        250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,        315, 320, 325 and 328 or more contiguous amino acids of 158P3D2        variant 1; the maximal lengths relevant for other variants are        shown in FIGS. 2A-2Q and 3A-3R respectively.

For example, representative embodiments of the invention disclosedherein include: polynucleotides and their encoded peptides themselvesencoding about amino acid 1 to about amino acid 10 of the 158P3D2protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 10 to about amino acid 20 of the 158P3D2 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 20 to about amino acid30 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 30 to about amino acid 40 of the 158P3D2protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 40 to about amino acid 50 of the 158P3D2 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 50 to about amino acid60 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 60 to about amino acid 70 of the 158P3D2protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 70 to about amino acid 80 of the 158P3D2 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 80 to about amino acid90 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 90 to about amino acid 100 of the 158P3D2protein shown in FIG. 2 or FIG. 3, in increments of about 10 aminoacids, ending at the carboxyl terminal amino acid set forth in FIG. 2 orFIG. 3. Accordingly, polynucleotides encoding portions of the amino acidsequence (of about 10 amino acids), of amino acids, 100 through thecarboxyl terminal amino acid of the 158P3D2 protein are embodiments ofthe invention. Wherein it is understood that each particular amino acidposition discloses that position plus or minus five amino acid residues.

Polynucleotides encoding relatively long portions of a 158P3D2 proteinare also within the scope of the invention. For example, polynucleotidesencoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about aminoacid 20, (or 30, or 40 or 50 etc.) of the 158P3D2 protein “or variant”shown in FIG. 2 or FIG. 3 can be generated by a variety of techniqueswell known in the art. These polynucleotide fragments can include anyportion of the 158P3D2 sequence as shown in FIG. 2.

Additional illustrative embodiments of the invention disclosed hereininclude 158P3D2 polynucleotide fragments encoding one or more of thebiological motifs contained within a 158P3D2 protein “or variant”sequence, including one or more of the motif-bearing subsequences of a158P3D2 protein “or variant” set forth in Tables VIII-XXI and XXII-XLIX.In another embodiment, typical polynucleotide fragments of the inventionencode one or more of the regions of 158P3D2 protein or variant thatexhibit homology to a known molecule. In another embodiment of theinvention, typical polynucleotide fragments can encode one or more ofthe 158P3D2 protein or variant N-glycosylation sites, cAMP andcGMP-dependent protein kinase phosphorylation sites, casein kinase IIphosphorylation sites or N-myristoylation site and amidation sites.

Note that to determine the starting position of any peptide set forth inTables VIII-XXI and Tables XXII to XLIX (collectively HLA PeptideTables) respective to its parental protein, e.g., variant 1, variant 2,etc., reference is made to three factors: the particular variant, thelength of the peptide in an HLA Peptide Table, and the Search Peptideslisted in Table VII. Generally, a unique Search Peptide is used toobtain HLA peptides for a particular variant. The position of eachSearch Peptide relative to its respective parent molecule is listed inTable VII. Accordingly, if a Search Peptide begins at position “X”, onemust add the value “X minus 1” to each position in Tables VIII-XXI andTables XXII-IL to obtain the actual position of the HLA peptides intheir parental molecule. For example if a particular Search Peptidebegins at position 150 of its parental molecule, one must add 150−1,i.e., 149 to each HLA peptide amino acid position to calculate theposition of that amino acid in the parent molecule.

II.A.) Uses of 158P3D2 Polynucleotides

II.A.1. Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. The human 158P3D2 gene maps to the chromosomallocation set forth in the Example entitled “Chromosomal Mapping of158P3D2.” For example, because the 158P3D2 gene maps to this chromosome,polynucleotides that encode different regions of the 158P3D2 proteinsare used to characterize cytogenetic abnormalities of this chromosomallocale, such as abnormalities that are identified as being associatedwith various cancers. In certain genes, a variety of chromosomalabnormalities including rearrangements have been identified as frequentcytogenetic abnormalities in a number of different cancers (see e.g.Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al.,Blood 86(10): 3905-3914 (1995) and Finger et al., P.N.A.S. 85(23):9158-9162 (1988)). Thus, polynucleotides encoding specific regions ofthe 158P3D2 proteins provide new tools that can be used to delineate,with greater precision than previously possible, cytogeneticabnormalities in the chromosomal region that encodes 158P3D2 that maycontribute to the malignant phenotype. In this context, thesepolynucleotides satisfy a need in the art for expanding the sensitivityof chromosomal screening in order to identify more subtle and lesscommon chromosomal abnormalities (see e.g. Evans et al., Am. J. Obstet.Gynecol 171(4): 1055-1057 (1994)).

Furthermore, as 158P3D2 was shown to be highly expressed in prostate andother cancers, 158P3D2 polynucleotides are used in methods assessing thestatus of 158P3D2 gene products in normal versus cancerous tissues.Typically, polynucleotides that encode specific regions of the 158P3D2proteins are used to assess the presence of perturbations (such asdeletions, insertions, point mutations, or alterations resulting in aloss of an antigen etc.) in specific regions of the 158P3D2 gene, suchas regions containing one or more motifs. Exemplary assays include bothRT-PCR assays as well as single-strand conformation polymorphism (SSCP)analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378(1999), both of which utilize polynucleotides encoding specific regionsof a protein to examine these regions within the protein.

II.A.2. Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of theinvention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone, or including alternative bases, whether derivedfrom natural sources or synthesized, and include molecules capable ofinhibiting the RNA or protein expression of 158P3D2. For example,antisense molecules can be RNAs or other molecules, including peptidenucleic acids (PNAs) or non-nucleic acid molecules such asphosphorothioate derivatives that specifically bind DNA or RNA in a basepair-dependent manner. A skilled artisan can readily obtain theseclasses of nucleic acid molecules using the 158P3D2 polynucleotides andpolynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,158P3D2. See for example, Jack Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5(1988). The 158P3D2 antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention can beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698(1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990).Additional 158P3D2 antisense oligonucleotides of the present inventioninclude morpholino antisense oligonucleotides known in the art (see,e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development6: 169-175).

The 158P3D2 antisense oligonucleotides of the present inventiontypically can be RNA or DNA that is complementary to and stablyhybridizes with the first 100 5′ codons or last 100 3′ codons of a158P3D2 genomic sequence or the corresponding mRNA. Absolutecomplementarity is not required, although high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to 158P3D2 mRNAand not to mRNA specifying other regulatory subunits of protein kinase.In one embodiment, 158P3D2 antisense oligonucleotides of the presentinvention are 15 to 30-mer fragments of the antisense DNA molecule thathave a sequence that hybridizes to 158P3D2 mRNA. Optionally, 158P3D2antisense oligonucleotide is a 30-mer oligonucleotide that iscomplementary to a region in the first 10 5′ codons or last 10 3′ codonsof 158P3D2. Alternatively, the antisense molecules are modified toemploy ribozymes in the inhibition of 158P3D2 expression, see, e.g., L.A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

II.A.3. Primers and Primer Pairs

Further specific embodiments of these nucleotides of the inventioninclude primers and primer pairs, which allow the specific amplificationof polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes can be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers are used todetect the presence of a 158P3D2 polynucleotide in a sample and as ameans for detecting a cell expressing a 158P3D2 protein.

Examples of such probes include polypeptides comprising all or part ofthe human 158P3D2 cDNA sequence shown in FIG. 2. Examples of primerpairs capable of specifically amplifying 158P3D2 mRNAs are alsodescribed in the Examples. As will be understood by the skilled artisan,a great many different primers and probes can be prepared based on thesequences provided herein and used effectively to amplify and/or detecta 158P3D2 mRNA.

The 158P3D2 polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the 158P3D2 gene(s), mRNA(s),or fragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as coding sequences capable ofdirecting the expression of 158P3D2 polypeptides; as tools formodulating or inhibiting the expression of the 158P3D2 gene(s) and/ortranslation of the 158P3D2 transcript(s); and as therapeutic agents.

The present invention includes the use of any probe as described hereinto identify and isolate a 158P3D2 or 158P3D2 related nucleic acidsequence from a naturally occurring source, such as humans or othermammals, as well as the isolated nucleic acid sequence per se, whichwould comprise all or most of the sequences found in the probe used.

II.A.4. Isolation of 158P3D2-Encoding Nucleic Acid Molecules

The 158P3D2 cDNA sequences described herein enable the isolation ofother polynucleotides encoding 158P3D2 gene product(s), as well as theisolation of polynucleotides encoding 158P3D2 gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms of a158P3D2 gene product as well as polynucleotides that encode analogs of158P3D2-related proteins. Various molecular cloning methods that can beemployed to isolate full length cDNAs encoding a 158P3D2 gene are wellknown (see, for example, Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989;Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley andSons, 1995). For example, lambda phage cloning methodologies can beconveniently employed, using commercially available cloning systems(e.g., Lambda ZAP Express, Stratagene). Phage clones containing 158P3D2gene cDNAs can be identified by probing with a labeled 158P3D2 cDNA or afragment thereof. For example, in one embodiment, a 158P3D2 cDNA (e.g.,FIG. 2) or a portion thereof can be synthesized and used as a probe toretrieve overlapping and full-length cDNAs corresponding to a 158P3D2gene. A 158P3D2 gene itself can be isolated by screening genomic DNAlibraries, bacterial artificial chromosome libraries (BACs), yeastartificial chromosome libraries (YACs), and the like, with 158P3D2 DNAprobes or primers.

II.A.5. Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containinga 158P3D2 polynucleotide, a fragment, analog or homologue thereof,including but not limited to phages, plasmids, phagemids, cosmids, YACs,BACs, as well as various viral and non-viral vectors well known in theart, and cells transformed or transfected with such recombinant DNA orRNA molecules. Methods for generating such molecules are Well known(see, for example, Sambrook et al., 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 158P3D2 polynucleotide, fragment,analog or homologue thereof within a suitable prokaryotic or eukaryotichost cell. Examples of suitable eukaryotic host cells include a yeastcell, a plant cell, or an animal cell, such as a mammalian cell or aninsect cell (e.g., a baculovirus-infectible cell such as an Sf9 orHighFive cell). Examples of suitable mammalian cells include variousprostate cancer cell lines such as DU145 and TsuPr1, other transfectableor transducible prostate cancer cell lines, primary cells (PrEC), aswell as a number of mammalian cells routinely used for the expression ofrecombinant proteins (e.g., COS, CHO, 293, 293T cells). Moreparticularly, a polynucleotide comprising the coding sequence of 158P3D2or a fragment, analog or homolog thereof can be used to generate 158P3D2proteins or fragments thereof using any number of host-vector systemsroutinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of158P3D2 proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, 158P3D2 can be expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-1, NIH3T3 and TsuPr1. The host-vector systems of the invention are useful forthe production of a 158P3D2 protein or fragment thereof. Suchhost-vector systems can be employed to study the functional propertiesof 158P3D2 and 158P3D2 mutations or analogs.

Recombinant human 158P3D2 protein or an analog or homolog or fragmentthereof can be produced by mammalian cells transfected with a constructencoding a 158P3D2-related nucleotide. For example, 293T cells can betransfected with an expression plasmid encoding 158P3D2 or fragment,analog or homolog thereof, a 158P3D2-related protein is expressed in the293T cells, and the recombinant 158P3D2 protein is isolated usingstandard purification methods (e.g., affinity purification usinganti-158P3D2 antibodies). In another embodiment, a 158P3D2 codingsequence is subcloned into the retroviral vector pSRαMSVtkneo and usedto infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 andrat-1 in order to establish 158P3D2 expressing cell lines. Various otherexpression systems well known in the art can also be employed.Expression constructs encoding a leader peptide joined in frame to a158P3D2 coding sequence can be used for the generation of a secretedform of recombinant 158P3D2 protein.

As discussed herein, redundancy in the genetic code permits variation in158P3D2 gene sequences. In particular, it is known in the art thatspecific host species often have specific codon preferences, and thusone can adapt the disclosed sequence as preferred for a desired host.For example, preferred analog codon sequences typically have rare codons(i.e., codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific species are calculated, for example, byutilizing codon usage tables available on the INTERNET such as at URLdna.affrc.go jp/˜nakamura/codon.html.

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080(1989). Skilled artisans understand that the general rule thateukaryotic ribosomes initiate translation exclusively at the 5′ proximalAUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

III.) 158P3D2-Related Proteins

Another aspect of the present invention provides 158P3D2-relatedproteins. Specific embodiments of 158P3D2 proteins comprise apolypeptide having all or part of the amino acid sequence of human158P3D2 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of158P3D2 proteins comprise variant, homolog or analog polypeptides thathave alterations in the amino acid sequence of 158P3D2 shown in FIG. 2or FIG. 3.

Embodiments of a 158P3D2 polypeptide include: a 158P3D2 polypeptidehaving a sequence shown in FIG. 2, a peptide sequence of a 158P3D2 asshown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of apolypeptide having the sequence as shown in FIG. 2; or, at least 10contiguous peptides of a polypeptide having the sequence as shown inFIG. 2 where T is U. For example, embodiments of 158P3D2 peptidescomprise, without limitation:

-   -   (I) a protein comprising, consisting essentially of, or        consisting of an amino acid sequence as shown in FIG. 2A-Q or        FIG. 3A-3R;    -   (II) a 158P3D2-related protein that is at least 90, 91, 92, 93,        94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino        acid sequence shown in FIG. 2A-Q or 3A-R;    -   (III) a 158P3D2-related protein that is at least 90, 91, 92, 93,        94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid        sequence shown in FIG. 2A-Q or 3A-R;    -   (IV) a protein that comprises at least one peptide set forth in        Tables VIII to XLIX, optionally with a proviso that it is not an        entire protein of FIG. 2;    -   (V) a protein that comprises at least one peptide set forth in        Tables VIII-XXI, collectively, which peptide is also set forth        in Tables XXII to XLIX, collectively, optionally with a proviso        that it is not an entire protein of FIG. 2;    -   (VI) a protein that comprises at least two peptides selected        from the peptides set forth in Tables VIII-XLIX, optionally with        a proviso that it is not an entire protein of FIG. 2;    -   (VII) a protein that comprises at least two peptides selected        from the peptides set forth in Tables VIII to XLIX collectively,        with a proviso that the protein is not a contiguous sequence        from an amino acid sequence of FIG. 2;    -   (VIII) a protein that comprises at least one peptide selected        from the peptides set forth in Tables VIII-XXI; and at least one        peptide selected from the peptides set forth in Tables XXII to        XLIX, with a proviso that the protein is not a contiguous        sequence from an amino acid sequence of FIG. 2;    -   (IX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG.        3A-3R in any whole number increment up to 328, 236, 181, 178,        1393, 1145, 1990, 2036, 610, 1978, and 2018 respectively that        includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35 amino acid position(s) having a value greater        than 0.5 in the Hydrophilicity profile of FIG. 5;    -   (X) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG.        3A-3R in any whole number increment up to 328, 236, 181, 178,        1393, 1145, 1990, 2036, 610, 1978, and 2018 respectively that        includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG.        3A-3R, in any whole number increment up to 328, 236, 181, 178,        1393, 1145, 1990, 2036, 610, 1978, and 2018 respectively that        includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value greater than 0.5 in the Percent Accessible Residues        profile of FIG. 7;    -   (XII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG.        3A-3R, in any whole number increment up to 328, 236, 181, 178,        1393, 1145, 1990, 2036, 610, 1978, and 2018 respectively that        includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value greater than 0.5 in the Average Flexibility profile of        FIG. 8;    -   (XIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG.        3A-3R in any whole number increment up to 328, 236, 181, 178,        1393, 1145, 1990, 2036, 610, 1978, and 2018 respectively that        includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a        value greater than 0.5 in the Beta-turn profile of FIG. 9;    -   (XIV) a peptide that occurs at least twice in Tables VIII-XXI        and XXII to XLIX, collectively;    -   (XV) a peptide that occurs at least three times in Tables        VIII-XXI and XXII to XLIX, collectively;    -   (XVI) a peptide that occurs at least four times in Tables        VIII-XXI and XXII to XLIX, collectively;    -   (XVII) a peptide that occurs at least five times in Tables        VIII-XXI and XXII to XLIX, collectively;    -   (XVIII) a peptide that occurs at least once in Tables VIII-XXI,        and at least once in tables XXII to XLIX;    -   (XIX) a peptide that occurs at least once in Tables VIII-XXI,        and at least twice in tables XXII to XLIX;    -   (XX) a peptide that occurs at least twice in Tables VIII-XXI,        and at least once in tables XXII to XLIX;    -   (XXI) a peptide that occurs at least twice in Tables VIII-XXI,        and at least twice in tables XXII to XLIX;    -   (XXII) a peptide which comprises one two, three, four, or five        of the following characteristics, or an oligonucleotide encoding        such peptide:    -   i) a region of at least 5 amino acids of a particular peptide of        FIG. 3, in any whole number increment up to the full length of        that protein in FIG. 3, that includes an amino acid position        having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9,        or having a value equal to 1.0, in the Hydrophilicity profile of        FIG. 5;    -   ii) a region of at least 5 amino acids of a particular peptide        of FIG. 3, in any whole number increment up to the full length        of that protein in FIG. 3, that includes an amino acid position        having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or        having a value equal to 0.0, in the Hydropathicity profile of        FIG. 6;    -   iii) a region of at least 5 amino acids of a particular peptide        of FIG. 3, in any whole number increment up to the full length        of that protein in FIG. 3, that includes an amino acid position        having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9,        or having a value equal to 1.0, in the Percent Accessible        Residues profile of FIG. 7;    -   iv) a region of at least 5 amino acids of a particular peptide        of FIG. 3, in any whole number increment up to the full length        of that protein in FIG. 3, that includes an amino acid position        having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9,        or having a value equal to 1.0, in the Average Flexibility        profile of FIG. 8; or,    -   v) a region of at least 5 amino acids of a particular peptide of        FIG. 3, in any whole number increment up to the full length of        that protein in FIG. 3, that includes an amino acid position        having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9,        or having a value equal to 1.0, in the Beta-turn profile of FIG.        9;    -   (XXIII) a composition comprising a peptide of (I)-(XXII) or an        antibody or binding region thereof together with a        pharmaceutical excipient and/or in a human unit dose form.    -   (XXIV) a method of using a peptide of (I)-(XXII), or an antibody        or binding region thereof or a composition of (XXIII) in a        method to modulate a cell expressing 158P3D2;    -   (XXV) a method of using a peptide of (I)-(XXII) or an antibody        or binding region thereof or a composition of (XXIII) in a        method to diagnose, prophylax, prognose, or treat an individual        who bears a cell expressing 158P3D2;    -   (XXVI) a method of using a peptide of (I)-(XXII) or an antibody        or binding region thereof or a composition (XXIII) in a method        to diagnose, prophylax, prognose, or treat an individual who        bears a cell expressing 158P3D2, said cell from a cancer of a        tissue listed in Table I;    -   (XXVII) a method of using a peptide of (I)-(XXII) or an antibody        or binding region thereof or a composition of (XXIII) in a        method to diagnose, prophylax, prognose, or treat a cancer;    -   (XXVIII) a method of using a peptide of (I)-(XXII) or an        antibody or binding region thereof or a composition of (XXIII)        in a method to diagnose, prophylax, prognose, or treat a a        cancer of a tissue listed in Table I;    -   (XXIX) a method of using a a peptide of (I)-(XXII) or an        antibody or binding region thereof or a composition and;    -   (XXIII) in a method to identify or characterize a modulator of a        cell expressing 158P3D2.

As used herein, a range is understood to specifically disclose all wholeunit positions thereof.

Typical embodiments of the invention disclosed herein include 158P3D2polynucleotides that encode specific portions of 158P3D2 mRNA sequences(and those which are complementary to such sequences) such as those thatencode the proteins and/or fragments thereof, for example:

-   -   (a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65,        70,75,80, 85,90,95, 100, 105, 110, 115, 120, 125, 130, 135, 140,        145; 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225,        250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,        315, 320, 325, and 328 or more contiguous amino acids of 158P3D2        variant 1; the maximal lengths relevant for other variants are        shown in FIGS. 2A-2Q and 3A-3R.

In general, naturally occurring allelic variants of human 158P3D2 sharea high degree of structural identity and homology (e.g., 90% or morehomology). Typically, allelic variants of a 158P3D2 protein containconservative amino acid substitutions within the 158P3D2 sequencesdescribed herein or contain a substitution of an amino acid from acorresponding position in a homologue of 158P3D2. One class of 158P3D2allelic variants are proteins that share a high degree of homology withat least a small region of a particular 158P3D2 amino acid sequence, butfurther contain a radical departure from the sequence, such as anon-conservative substitution, truncation, insertion or frame shift. Incomparisons of protein sequences, the terms, similarity, identity, andhomology each have a distinct meaning as appreciated in the field ofgenetics. Moreover, orthology and paralogy can be important conceptsdescribing the relationship of members of a given protein family in oneorganism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative aminoacid substitutions can frequently be made in a protein without alteringeither the conformation or the function of the protein. Proteins of theinvention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15conservative substitutions. Such changes include substituting any ofisoleucine (I), valine (V), and leucine (L) for any other of thesehydrophobic amino acids; aspartic acid (D) for glutamic acid (E) andvice versa; glutamine (Q) for asparagine (N) and vice versa; and serine(S) for threonine (T) and vice versa. Other substitutions can also beconsidered conservative, depending on the environment of the particularamino acid and its role in the three-dimensional structure of theprotein. For example, glycine (G) and alanine (A) can frequently beinterchangeable, as can alanine (A) and valine (V). Methionine (M),which is relatively hydrophobic, can frequently be interchanged withleucine and isoleucine, and sometimes with valine. Lysine (K) andarginine (R) are frequently interchangeable in locations in which thesignificant feature of the amino acid residue is its charge and thediffering pK's of these two amino acid residues are not significant.Still other changes can be considered “conservative” in particularenvironments (see, e.g. Table III herein; pages 13-15 “Biochemistry” 2ndED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19;270(20):11882-6).

Embodiments of the invention disclosed herein include a wide variety ofart-accepted variants or analogs of 158P3D2 proteins such aspolypeptides having amino acid insertions, deletions and substitutions.158P3D2 variants can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassettemutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selectionmutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415(1986)) or other known techniques can be performed on the cloned DNA toproduce the 158P3D2 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence that is involved in aspecific biological activity such as a protein-protein interaction.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia,J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yieldadequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 158P3D2 variants, analogs or homologs, have thedistinguishing attribute of having at least one epitope that is “crossreactive” with a 158P3D2 protein having an amino acid sequence of FIG.3. As used in this sentence, “cross reactive” means that an antibody orT cell that specifically binds to a 158P3D2 variant also specificallybinds to a 158P3D2 protein having an amino acid sequence set forth inFIG. 3. A polypeptide ceases to be a variant of a protein shown in FIG.3, when it no longer contains any epitope capable of being recognized byan antibody or T cell that specifically binds to the starting 158P3D2protein. Those skilled in the art understand that antibodies thatrecognize proteins bind to epitopes of varying size, and a grouping ofthe order of about four or five amino acids, contiguous or not, isregarded as a typical number of amino acids in a minimal epitope. See,e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955; Hebbes et al.,Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985)135(4):2598-608.

Other classes of 158P3D2-related protein variants share 70%, 75%, 80%,85% or 90% or more similarity with an amino acid sequence of FIG. 3, ora fragment thereof. Another specific class of 158P3D2 protein variantsor analogs comprises one or more of the 158P3D2 biological motifsdescribed herein or presently known in the art. Thus, encompassed by thepresent invention are analogs of 158P3D2 fragments (nucleic or aminoacid) that have altered functional (e.g. immunogenic) propertiesrelative to the starting fragment. It is to be appreciated that motifsnow or which become part of the art are to be applied to the nucleic oramino acid sequences of FIG. 2 or FIG. 3.

As discussed herein, embodiments of the claimed invention includepolypeptides containing less than the full amino acid sequence of a158P3D2 protein shown in FIG. 2 or FIG. 3. For example, representativeembodiments of the invention comprise peptides/proteins having any 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a158P3D2 protein shown in FIG. 2 or FIG. 3.

Moreover, representative embodiments of the invention disclosed hereininclude polypeptides consisting of about amino acid 1 to about aminoacid 10 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 10 to about amino acid 20 of a 158P3D2protein shown in FIG. 2 or FIG. 3, polypeptides consisting of aboutamino acid 20 to about amino acid 30 of a 158P3D2 protein shown in FIG.2 or FIG. 3, polypeptides consisting of about amino acid 30 to aboutamino acid 40 of a 158P3D2 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 40 to about amino acid 50 ofa 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 50 to about amino acid 60 of a 158P3D2 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 toabout amino acid 70 of a 158P3D2 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 70 to about amino acid 80 ofa 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 80 to about amino acid 90 of a 158P3D2 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 toabout amino acid 100 of a 158P3D2 protein shown in FIG. 2 or FIG. 3,etc. throughout the entirety of a 158P3D2 amino acid sequence. Moreover,polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.)to about amino acid 20, (or 130, or 140 or 150 etc.) of a 158P3D2protein shown in FIG. 2 or FIG. 3 are embodiments of the invention. Itis to be appreciated that the starting and stopping positions in thisparagraph refer to the specified position as well as that position plusor minus 5 residues. 158P3D2-related proteins are generated usingstandard peptide synthesis technology or using chemical cleavage methodswell known in the art. Alternatively, recombinant methods can be used togenerate nucleic acid molecules that encode a 158P3D2-related protein.In one embodiment, nucleic acid molecules provide a means to generatedefined fragments of a 158P3D2 protein (or variants, homologs or analogsthereof).

III.A.) Motif-Bearing Protein Embodiments

Additional illustrative embodiments of the invention disclosed hereininclude 158P3D2 polypeptides comprising the amino acid residues of oneor more of the biological motifs contained within a 158P3D2 polypeptidesequence set forth in FIG. 2 or FIG. 3. Various motifs are known in theart, and a protein can be evaluated for the presence of such motifs by anumber of publicly available Internet sites (see, e.g., URL addresses:pfam.wustl.edu/;searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html;psort.ims.u-tokyo.acjp/; cbs.dtu.dk/; ebi.ac.uk/interpro/scan.html;expasy.ch/tools/scnpsit1.html; Epimatrix™ and Epimer™, Brown University,brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS,bimas.dcrt.nih.gov/.).

Motif bearing subsequences of all 158P3D2 variant proteins are set forthand identified in Tables VIII-XXI and XXII-XLIX.

Table V sets forth several frequently occurring motifs based on pfamsearches (see URL address pfam.wustl.edu/). The columns of Table V list(1) motif name abbreviation, (2) percent identity found amongst thedifferent member of the motif family, (3) motif name or description and(4) most common function; location information is included if the motifis relevant for location.

Polypeptides comprising one or more of the 158P3D2 motifs discussedabove are useful in elucidating the specific characteristics of amalignant phenotype in view of the observation that the 158P3D2 motifsdiscussed above are associated with growth dysregulation and because158P3D2 is overexpressed in certain cancers (See, e.g., Table I). Caseinkinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C,for example, are enzymes known to be associated with the development ofthe malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2):165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995);Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterzielet al., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2):305-309 (1998)). Moreover, both glycosylation and myristoylation areprotein modifications also associated with cancer and cancer progression(see e.g. Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999);Raju et al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation isanother protein modification also associated with cancer and cancerprogression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr.(13): 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more ofthe immunoreactive epitopes identified in accordance with art-acceptedmethods, such as the peptides set forth in Tables VIII-XXI andXXII-XLIX. CTL epitopes can be determined using specific algorithms toidentify peptides within a 158P3D2 protein that are capable of optimallybinding to specified HLA alleles (e.g., Table IV; Epimatrix™ andEpimer™, Brown University, URLbrown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, URLbimas.dcrt.nih.gov/.) Moreover, processes for identifying peptides thathave sufficient binding affinity for HLA molecules and which arecorrelated with being immunogenic epitopes, are well known in the art,and are carried out without undue experimentation. In addition,processes for identifying peptides that are immunogenic epitopes, arewell known in the art, and are carried out without undue experimentationeither in vitro or in vivo.

Also known in the art are principles for creating analogs of suchepitopes in order to modulate immunogenicity. For example, one beginswith an epitope that bears a CTL or HTL motif (see, e.g., the HLA ClassI and HLA Class II motifs/supermotifs of Table IV). The epitope isanaloged by substituting out an amino acid at one of the specifiedpositions, and replacing it with another amino acid specified for thatposition. For example, on the basis of residues defined in Table IV, onecan substitute out a deleterious residue in favor of any other residue,such as a preferred residue; substitute a less-preferred residue with apreferred residue; or substitute an originally-occurring preferredresidue with another preferred residue. Substitutions can occur atprimary anchor positions or at other positions in a peptide; see, e.g.,Table IV.

A variety of references reflect the art regarding the identification andgeneration of epitopes in a protein of interest as well as analogsthereof. See, for example, WO 97/33602 to Chesnut et al.; Sette,Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondoet al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol.1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt etal., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7(1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3):266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633;Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J.Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9):751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.

Related embodiments of the invention include polypeptides comprisingcombinations of the different motifs set forth in Table VI, and/or, oneor more of the predicted CTL epitopes of Tables VIII-XXI and XXII-XLIX,and/or, one or more of the predicted HTL epitopes of Tables XLVI-XLIX,and/or, one or more of the T cell binding motifs known in the art.Preferred embodiments contain no insertions, deletions or substitutionseither within the motifs or within the intervening sequences of thepolypeptides. In addition, embodiments which include a number of eitherN-terminal and/or C-terminal amino acid residues on either side of thesemotifs may be desirable (to, for example, include a greater portion ofthe polypeptide architecture in which the motif is located). Typically,the number of N-terminal and/or C-terminal amino acid residues on eitherside of a motif is between about 1 to about 100 amino acid residues,preferably 5 to about 50 amino acid residues.

158P3D2-related proteins are embodied in many forms, preferably inisolated form. A purified 158P3D2 protein molecule will be substantiallyfree of other proteins or molecules that impair the binding of 158P3D2to antibody, T cell or other ligand. The nature and degree of isolationand purification will depend on the intended use. Embodiments of a158P3D2-related proteins include purified 158P3D2-related proteins andfunctional, soluble 158P3D2-related proteins. In one embodiment, afunctional, soluble 158P3D2 protein or fragment thereof retains theability to be bound by antibody, T cell or other ligand.

The invention also provides 158P3D2 proteins comprising biologicallyactive fragments of a 158P3D2 amino acid sequence shown in FIG. 2 orFIG. 3. Such proteins exhibit properties of the starting 158P3D2protein, such as the ability to elicit the generation of antibodies thatspecifically bind an epitope associated with the starting 158P3D2protein; to be bound by such antibodies; to elicit the activation of HTLor CTL; and/or, to be recognized by HTL or CTL that also specificallybind to the starting protein.

158P3D2-related polypeptides that contain particularly interestingstructures can be predicted and/or identified using various analyticaltechniques well known in the art, including, for example, the methods ofChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis, or based on immunogenicity. Fragments thatcontain such structures are particularly useful in generatingsubunit-specific anti-158P3D2 antibodies or T cells or in identifyingcellular factors that bind to 158P3D2. For example, hydrophilicityprofiles can be generated, and immunogenic peptide fragments identified,using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can begenerated, and immunogenic peptide fragments identified, using themethod of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol.157:105-132. Percent (%) Accessible Residues profiles can be generated,and immunogenic peptide fragments identified, using the method of JaninJ., 1979, Nature 277:491-492. Average Flexibility profiles can begenerated, and immunogenic peptide fragments identified, using themethod of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. ProteinRes. 32:242-255. Beta-turn profiles can be generated, and immunogenicpeptide fragments identified, using the method of Deleage, G., Roux B.,1987, Protein Engineering 1:289-294.

CTL epitopes can be determined using specific algorithms to identifypeptides within a 158P3D2 protein that are capable of optimally bindingto specified HLA alleles (e.g., by using the SYFPEITHI site at WorldWide Web URL syfpeithi.bmi-heidelberg.com/; the listings in TableIV(A)-(E); Epimatrix™ and Epimer™, Brown University, URL(brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and BIMAS, URLbimas.dcrt.nih.gov/). Illustrating this, peptide epitopes from 158P3D2that are presented in the context of human MHC Class I molecules, e.g.,HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (see, e.g., TablesVIII-XXI, XXII-XLIX). Specifically, the complete amino acid sequence ofthe 158P3D2 protein and relevant portions of other variants, i.e., forHLA Class I predictions 9 flanking residues on either side of a pointmutation or exon juction, and for HLA Class II predictions 14 flankingresidues on either side of a point mutation or exon junctioncorresponding to that variant, were entered into the HLA Peptide MotifSearch algorithm found in the Bioinformatics and Molecular AnalysisSection (BIMAS) web site listed above; in addition to the siteSYFPEITHI, at URL syfpeithi.bmi-heidelberg.com/.

The HLA peptide motif search algorithm was developed by Dr. Ken Parkerbased on binding of specific peptide sequences in the groove of HLAClass I molecules, in particular HLA-A2 (see, e.g., Falk et al., Nature351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker etal., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol.152:163-75 (1994)). This algorithm allows location and ranking of 8-mer,9-mer, and 10-mer peptides from a complete protein sequence forpredicted binding to HLA-A2 as well as numerous other HLA Class Imolecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers.For example, for Class I HLA-A2, the epitopes preferably contain aleucine (L) or methionine (M) at position 2 and a valine (V) or leucine(L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7(1992)). Selected results of 158P3D2 predicted binding peptides areshown in Tables VIII-XXI and XXII-XLIX herein. In Tables VIII-XXI andXXII-XLVII, selected candidates, 9-mers and 10-mers, for each familymember are shown along with their location, the amino acid sequence ofeach specific peptide, and an estimated binding score. In TablesXLVI-XLIX, selected candidates, 15-mers, for each family member areshown along with their location, the amino acid sequence of eachspecific peptide, and an estimated binding score. The binding scorecorresponds to the estimated half time of dissociation of complexescontaining the peptide at 37° C. at pH 6.5. Peptides with the highestbinding score are predicted to be the most tightly bound to HLA Class Ion the cell surface for the greatest period of time and thus representthe best immunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated bystabilization of HLA expression on the antigen-processing defective cellline T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa etal., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides canbe evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes(CTL) in the presence of antigen presenting cells such as dendriticcells.

It is to be appreciated that every epitope predicted by the BIMAS site,Epimer™ and Epimatrix™ sites, or specified by the HLA class I or classII motifs available in the art or which become part of the art such asset forth in Table IV (or determined using World Wide Web site URLsyfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) are to be“applied” to a 158P3D2 protein in accordance with the invention. As usedin this context “applied” means that a 158P3D2 protein is evaluated,e.g., visually or by computer-based patterns finding methods, asappreciated by those of skill in the relevant art. Every subsequence ofa 158P3D2 protein of 8, 9, 10, or 11 amino acid residues that bears anHLA Class I motif, or a subsequence of 9 or more amino acid residuesthat bear an HLA Class II motif are within the scope of the invention.

III.B.) Expression of 158P3D2-Related Proteins

In an embodiment described in the examples that follow, 158P3D2 can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding 158P3D2 with a C-terminal 6×His and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTenn.). The Tag5 vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted 158P3D2 protein intransfected cells. The secreted HIS-tagged 158P3D2 in the culture mediacan be purified, e.g., using a nickel column using standard techniques.

III.C.) Modifications of 158P3D2-Related Proteins

Modifications of 158P3D2-related proteins such as covalent modificationsare included within the scope of this invention. One type of covalentmodification includes reacting targeted amino acid residues of a 158P3D2polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues of a158P3D2 protein. Another type of covalent modification of a 158P3D2polypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of a protein of the invention.Another type of covalent modification of 158P3D2 comprises linking a158P3D2 polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 158P3D2-related proteins of the present invention can also bemodified to form a chimeric molecule comprising 158P3D2 fused toanother, heterologous polypeptide or amino acid sequence. Such achimeric molecule can be synthesized chemically or recombinantly. Achimeric molecule can have a protein of the invention fused to anothertumor-associated antigen or fragment thereof. Alternatively, a proteinin accordance with the invention can comprise a fusion of fragments of a158P3D2 sequence (amino or nucleic acid) such that a molecule is createdthat is not, through its length, directly homologous to the amino ornucleic acid sequences shown in FIG. 2 or FIG. 3. Such a chimericmolecule can comprise multiples of the same subsequence of 158P3D2. Achimeric molecule can comprise a fusion of a 158P3D2-related proteinwith a polyhistidine epitope tag, which provides an epitope to whichimmobilized nickel can selectively bind, with cytokines or with growthfactors. The epitope tag is generally placed at the amino- orcarboxyl-terminus of a 158P3D2 protein. In an alternative embodiment,the chimeric molecule can comprise a fusion of a 158P3D2-related proteinwith an immunoglobulin or a particular region of an immunoglobulin. Fora bivalent form of the chimeric molecule (also referred to as an“immunoadhesin”), such a fusion could be to the Fc region of an IgGmolecule. The Ig fusions preferably include the substitution of asoluble (transmembrane domain deleted or inactivated) form of a 158P3D2polypeptide in place of at least one variable region within an Igmolecule. In a preferred embodiment, the immunoglobulin fusion includesthe hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of anIgG1 molecule. For the production of immunoglobulin fusions see, e.g.,U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

III.D.) Uses of 158P3D2-Related Proteins

The proteins of the invention have a number of different specific uses.As 158P3D2 is highly expressed in prostate and other cancers,158P3D2-related proteins are used in methods that assess the status of158P3D2 gene products in normal versus cancerous tissues, therebyelucidating the malignant phenotype. Typically, polypeptides fromspecific regions of a 158P3D2 protein are used to assess the presence ofperturbations (such as deletions, insertions, point mutations etc.) inthose regions (such as regions containing one or more motifs). Exemplaryassays utilize antibodies or T cells targeting 158P3D2-related proteinscomprising the amino acid residues of one or more of the biologicalmotifs contained within a 158P3D2 polypeptide sequence in order toevaluate the characteristics of this region in normal versus canceroustissues or to elicit an immune response to the epitope. Alternatively,158P3D2-related proteins that contain the amino acid residues of one ormore of the biological motifs in a 158P3D2 protein are used to screenfor factors that interact with that region of 158P3D2.

158P3D2 protein fragments/subsequences are particularly useful ingenerating and characterizing domain-specific antibodies (e.g.,antibodies recognizing an extracellular or intracellular epitope of a158P3D2 protein), for identifying agents or cellular factors that bindto 158P3D2 or a particular structural domain thereof, and in varioustherapeutic and diagnostic contexts, including but not limited todiagnostic assays, cancer vaccines and methods of preparing suchvaccines.

Proteins encoded by the 158P3D2 genes, or by analogs, homologs orfragments thereof, have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to a 158P3D2 gene product.Antibodies raised against a 158P3D2 protein or fragment thereof areuseful in diagnostic and prognostic assays, and imaging methodologies inthe management of human cancers characterized by expression of 158P3D2protein, such as those listed in Table I. Such antibodies can beexpressed intracellularly and used in methods of treating patients withsuch cancers. 158P3D2-related nucleic acids or proteins are also used ingenerating HTL or CTL responses.

Various immunological assays useful for the detection of 158P3D2proteins are used, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Antibodies can be labeled and used asimmunological imaging reagents capable of detecting 158P3D2-expressingcells (e.g., in radioscintigraphic imaging methods). 158P3D2 proteinsare also particularly useful in generating cancer vaccines, as furtherdescribed herein.

IV.) 158P3D2 Antibodies

Another aspect of the invention provides antibodies that bind to158P3D2-related proteins. Preferred antibodies specifically bind to a158P3D2-related protein and do not bind (or bind weakly) to peptides orproteins that are not 158P3D2-related proteins under physiologicalconditions. In this context, examples of physiological conditionsinclude: 1) phosphate buffered saline; 2) Tris-buffered salinecontaining 25 mM Tris and 150 mM NaCl; or normal saline (0.9% NaCl); 4)animal serum such as human serum; or, 5) a combination of any of 1)through 4); these reactions preferably taking place at pH 7.5,alternatively in a range of pH 7.0 to 8.0, or alternatively in a rangeof pH 6.5 to 8.5; also, these reactions taking place at a temperaturebetween 4° C. to 37° C. For example, antibodies that bind 158P3D2 canbind 158P3D2-related proteins such as the homologs or analogs thereof.

158P3D2 antibodies of the invention are particularly useful in cancer(see, e.g., Table I) diagnostic and prognostic assays, and imagingmethodologies. Similarly, such antibodies are useful in the treatment,diagnosis, and/or prognosis of other cancers, to the extent 158P3D2 isalso expressed or overexpressed in these other cancers. Moreover,intracellularly expressed antibodies (e.g., single chain antibodies) aretherapeutically useful in treating cancers in which the expression of158P3D2 is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for thedetection and quantification of 158P3D2 and mutant 158P3D2-relatedproteins. Such assays can comprise one or more 158P3D2 antibodiescapable of recognizing and binding a 158P3D2-related protein, asappropriate. These assays are performed within various immunologicalassay formats well known in the art, including but not limited tovarious types of radioimmunoassays, enzyme-linked immunosorbent assays(ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cellimmunogenicity assays (inhibitory or stimulatory) as well as majorhistocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostatecancer and other cancers expressing 158P3D2 are also provided by theinvention, including but not limited to radioscintigraphic imagingmethods using labeled 158P3D2 antibodies. Such assays are clinicallyuseful in the detection, monitoring, and prognosis of 158P3D2 expressingcancers such as prostate cancer.

158P3D2 antibodies are also used in methods for purifying a158P3D2-related protein and for isolating 158P3D2 homologues and relatedmolecules. For example, a method of purifying a 158P3D2-related proteincomprises incubating a 158P3D2 antibody, which has been coupled to asolid matrix, with a lysate or other solution containing a158P3D2-related protein under conditions that permit the 158P3D2antibody to bind to the 158P3D2-related protein; washing the solidmatrix to eliminate impurities; and eluting the 158P3D2-related proteinfrom the coupled antibody. Other uses of 158P3D2 antibodies inaccordance with the invention include generating anti-idiotypicantibodies that mimic a 158P3D2 protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies can be prepared by immunizing a suitablemammalian host using a 158P3D2-related protein, peptide, or fragment, inisolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSHPress, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold SpringHarbor Press, NY (1989)). In addition, fusion proteins of 158P3D2 canalso be used, such as a 158P3D2 GST-fusion protein. In a particularembodiment, a GST fusion protein comprising all or most of the aminoacid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogento generate appropriate antibodies. In another embodiment, a158P3D2-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified 158P3D2-related protein or 158P3D2 expressingcells) to generate an immune response to the encoded immunogen (forreview, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of a 158P3D2 protein as shown in FIG. 2 or FIG.3 can be analyzed to select specific regions of the 158P3D2 protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of a 158P3D2 amino acid sequence are used to identifyhydrophilic regions in the 158P3D2 structure. Regions of a 158P3D2protein that show immunogenic structure, as well as other regions anddomains, can readily be identified using various other methods known inthe art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can begenerated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc.Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can begenerated using the method of Kyte, J. and Doolittle, R. F., 1982, J.Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can begenerated using the method of Janin J., 1979, Nature 277:491-492.Average Flexibility profiles can be generated using the method ofBhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res.32:242-255. Beta-turn profiles can be generated using the method ofDeleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, eachregion identified by any of these programs or methods is within thescope of the present invention. Methods for the generation of 158P3D2antibodies are further illustrated by way of the examples providedherein. Methods for preparing a protein or polypeptide for use as animmunogen are well known in the art. Also well known in the art aremethods for preparing immunogenic conjugates of a protein with acarrier, such as BSA, KLH or other carrier protein. In somecircumstances, direct conjugation using, for example, carbodiimidereagents are used; in other instances linking reagents such as thosesupplied by Pierce Chemical Co., Rockford, Ill., are effective.Administration of a 158P3D2 immunogen is often conducted by injectionover a suitable time period and with use of a suitable adjuvant, as isunderstood in the art. During the immunization schedule, titers ofantibodies can be taken to determine adequacy of antibody formation.

158P3D2 monoclonal antibodies can be produced by various means wellknown in the art. For example, immortalized cell lines that secrete adesired monoclonal antibody are prepared using the standard hybridomatechnology of Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known. Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a 158P3D2-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, byrecombinant means. Regions that bind specifically to the desired regionsof a 158P3D2 protein can also be produced in the context of chimeric orcomplementarity-determining region (CDR) grafted antibodies of multiplespecies origin. Humanized or human 158P3D2 antibodies can also beproduced, and are preferred for use in therapeutic contexts. Methods forhumanizing murine and other non-human antibodies, by substituting one ormore of the non-human antibody CDRs for corresponding human antibodysequences, are well known (see for example, Jones et al., 1986, Nature321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen etal., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc.Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151:2296.

Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan et al., 1998,Nature Biotechnology 16: 535-539). Fully human 158P3D2 monoclonalantibodies can be generated using cloning technologies employing largehuman Ig gene combinatorial libraries (i.e., phage display) (Griffithsand Hoogenboom, Building an in vitro immune system: human antibodiesfrom phage display libraries. In: Protein Engineering of AntibodyMolecules for Prophylactic and Therapeutic Applications in Man, Clark,M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, HumanAntibodies from combinatorial libraries. Id., pp 65-82). Fully human158P3D2 monoclonal antibodies can also be produced using transgenic miceengineered to contain human immunoglobulin gene loci as described in PCTPatent Application WO98/24893, Kucherlapati and Jakobovits et al.,published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest.Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued 19 Dec. 2000; U.S.Pat. No. 6,150,584 issued 12 Nov. 2000; and, U.S. Pat. No. 6,114,598issued 5 Sep. 2000). This method avoids the in vitro manipulationrequired with phage display technology and efficiently produces highaffinity authentic human antibodies.

Reactivity of 158P3D2 antibodies with a 158P3D2-related protein can beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,158P3D2-related proteins, 158P3D2-expressing cells or extracts thereof.A 158P3D2 antibody or fragment thereof can be labeled with a detectablemarker or conjugated to a second molecule. Suitable detectable markersinclude, but are not limited to, a radioisotope, a fluorescent compound,a bioluminescent compound, chemiluminescent compound, a metal chelatoror an enzyme. Further, bi-specific antibodies specific for two or more158P3D2 epitopes are generated using methods generally known in the art.Homodimeric antibodies can also be generated by cross-linking techniquesknown in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

V.) 158P3D2 Cellular Immune Responses

The mechanism by which T cells recognize antigens has been delineated.

Efficacious peptide epitope vaccine compositions of the invention inducea therapeutic or prophylactic immune responses in very broad segments ofthe world-wide population. For an understanding of the value andefficacy of compositions of the invention that induce cellular immuneresponses, a brief review of immunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligandrecognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071,1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. andBodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev.Immunol. 11:403, 1993). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs required for specific binding to HLA antigen molecules have beenidentified and are set forth in Table IV (see also, e.g., Southwood, etal., J.

Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995;Rammensee et al., SYFPEITHI, access via World Wide Web at URL(134.2.96.221/scripts.hlaserver.dll/home.htm); Sette, A. and Sidney, J.Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin.Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol.4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994;Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol.155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996;Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J.Immunogenetics 1999 November; 50(3-4):201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexeshave revealed pockets within the peptide binding cleft/groove of HLAmolecules which accommodate, in an allele-specific mode, residues borneby peptide ligands; these residues in turn determine the HLA bindingcapacity of the peptides in which they are present. (See, e.g., Madden,D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203,1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H.et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci.USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M.L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927,1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D.C., J. Mol.Biol. 219:277, 1991.)

Accordingly, the definition of class I and class II allele-specific HLAbinding motifs, or class I or class II supermotifs allows identificationof regions within a protein that are correlated with binding toparticular HLA antigen(s).

Thus, by a process of HLA motif identification, candidates forepitope-based vaccines have been identified; such candidates can befurther evaluated by HLA-peptide binding assays to determine bindingaffinity and/or the time period of association of the epitope and itscorresponding HLA molecule. Additional confirmatory work can beperformed to select, amongst these vaccine candidates, epitopes withpreferred characteristics in terms of population coverage, and/orimmunogenicity.

Various strategies can be utilized to evaluate cellular immunogenicity,including:

-   -   1) Evaluation of primary T cell cultures from normal individuals        (see, e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995;        Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994;        Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et        al., Human Immunol. 59:1, 1998). This procedure involves the        stimulation of peripheral blood lymphocytes (PBL) from normal        subjects with a test peptide in the presence of antigen        presenting cells in vitro over a period of several weeks. T        cells specific for the peptide become activated during this time        and are detected using, e.g., a lymphokine- or 51Cr-release        assay involving peptide sensitized target cells.    -   2) Immunization of HLA transgenic mice (see, e.g.,        Wentworth, P. A. et al., J. Immunol. 26:97, 1996;        Wentworth, P. A. et al., Int. Immunol. 8:651, 1996;        Alexander, J. et al., J. Immunol. 159:4753, 1997). For example,        in such methods peptides in incomplete Freund's adjuvant are        administered subcutaneously to HLA transgenic mice. Several        weeks following immunization, splenocytes are removed and        cultured in vitro in the presence of test peptide for        approximately one week. Peptide-specific T cells are detected        using, e.g., a 51Cr-release assay involving peptide sensitized        target cells and target cells expressing endogenously generated        antigen.    -   3) Demonstration of recall T cell responses from immune        individuals who have been either effectively vaccinated and/or        from chronically ill patients (see, e.g., Rehermann, B. et        al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et al., Immunity        7:97, 1997; Bertoni, R. et al., J. Clin. Invest. 100:503, 1997;        Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997;        Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly,        recall responses are detected by culturing PBL from subjects        that have been exposed to the antigen due to disease and thus        have generated an immune response “naturally”, or from patients        who were vaccinated against the antigen. PBL from subjects are        cultured in vitro for 1-2 weeks in the presence of test peptide        plus antigen presenting cells (APC) to allow activation of        “memory” T cells, as compared to “naive” T cells. At the end of        the culture period, T cell activity is detected using assays        including 51 Cr release involving peptide-sensitized targets, T        cell proliferation, or lymphokine release.        VI.) 158P3D2 Transgenic Animals

Nucleic acids that encode a 158P3D2-related protein can also be used togenerate either transgenic animals or “knock out” animals that, in turn,are useful in the development and screening of therapeutically usefulreagents. In accordance with established techniques, cDNA encoding158P3D2 can be used to clone genomic DNA that encodes 158P3D2. Thecloned genomic sequences can then be used to generate transgenic animalscontaining cells that express DNA that encode 158P3D2. Methods forgenerating transgenic animals, particularly animals such as mice orrats, have become conventional in the art and are described, forexample, in U.S. Pat. No. 4,736,866 issued 12 Apr. 1988, and U.S. Pat.No. 4,870,009 issued 26 Sep. 1989. Typically, particular cells would betargeted for 158P3D2 transgene incorporation with tissue-specificenhancers.

Transgenic animals that include a copy of a transgene encoding 158P3D2can be used to examine the effect of increased expression of DNA thatencodes 158P3D2. Such animals can be used as tester animals for reagentsthought to confer protection from, for example, pathological conditionsassociated with its overexpression. In accordance with this aspect ofthe invention, an animal is treated with a reagent and a reducedincidence of a pathological condition, compared to untreated animalsthat bear the transgene, would indicate a potential therapeuticintervention for the pathological condition.

Alternatively, non-human homologues of 158P3D2 can be used to constructa 158P3D2 “knock out” animal that has a defective or altered geneencoding 158P3D2 as a result of homologous recombination between theendogenous gene encoding 158P3D2 and altered genomic DNA encoding158P3D2 introduced into an embryonic cell of the animal. For example,cDNA that encodes 158P3D2 can be used to clone genomic DNA encoding158P3D2 in accordance with established techniques. A portion of thegenomic DNA encoding 158P3D2 can be deleted or replaced with anothergene, such as a gene encoding a selectable marker that can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras (see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152). A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal, and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knock out animals can becharacterized, for example, for their ability to defend against certainpathological conditions or for their development of pathologicalconditions due to absence of a 158P3D2 polypeptide.

VII.) Methods for the Detection of 158P3D2

Another aspect of the present invention relates to methods for detecting158P3D2 polynucleotides and 158P3D2-related proteins, as well as methodsfor identifying a cell that expresses 158P3D2. The expression profile of158P3D2 makes it a diagnostic marker for metastasized disease.Accordingly, the status of 158P3D2 gene products provides informationuseful for predicting a variety of factors including susceptibility toadvanced stage disease, rate of progression, and/or tumoraggressiveness. As discussed in detail herein, the status of 158P3D2gene products in patient samples can be analyzed by a variety protocolsthat are well known in the art including immunohistochemical analysis,the variety of Northern blotting techniques including in situhybridization, RT-PCR analysis (for example on laser capturemicro-dissected samples), Western blot analysis and tissue arrayanalysis.

More particularly, the invention provides assays for the detection of158P3D2 polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable 158P3D2 polynucleotides include, for example, a 158P3D2gene or fragment thereof, 158P3D2 mRNA, alternative splice variant158P3D2 mRNAs, and recombinant DNA or RNA molecules that contain a158P3D2 polynucleotide. A number of methods for amplifying and/ordetecting the presence of 158P3D2 polynucleotides are well known in theart and can be employed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a 158P3D2 mRNA in a biologicalsample comprises producing cDNA from the sample by reverse transcriptionusing at least one primer; amplifying the cDNA so produced using a158P3D2 polynucleotides as sense and antisense primers to amplify158P3D2 cDNAs therein; and detecting the presence of the amplified158P3D2 cDNA. Optionally, the sequence of the amplified 158P3D2 cDNA canbe determined.

In another embodiment, a method of detecting a 158P3D2 gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using 158P3D2 polynucleotides assense and antisense primers; and detecting the presence of the amplified158P3D2 gene. Any number of appropriate sense and antisense probecombinations can be designed from a 158P3D2 nucleotide sequence (see,e.g., FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of a158P3D2 protein in a tissue or other biological sample such as serum,semen, bone, prostate, urine, cell preparations, and the like. Methodsfor detecting a 158P3D2-related protein are also well known and include,for example, immunoprecipitation, immunohistochemical analysis, Westernblot analysis, molecular binding assays, ELISA, ELIFA and the like. Forexample, a method of detecting the presence of a 158P3D2-related proteinin a biological sample comprises first contacting the sample with a158P3D2 antibody, a 158P3D2-reactive fragment thereof, or a recombinantprotein containing an antigen-binding region of a 158P3D2 antibody; andthen detecting the binding of 158P3D2-related protein in the sample.

Methods for identifying a cell that expresses 158P3D2 are also withinthe scope of the invention. In one embodiment, an assay for identifyinga cell that expresses a 158P3D2 gene comprises detecting the presence of158P3D2 mRNA in the cell. Methods for the detection of particular mRNAsin cells are well known and include, for example, hybridization assaysusing complementary DNA probes (such as in situ hybridization usinglabeled 158P3D2 riboprobes, Northern blot and related techniques) andvarious nucleic acid amplification assays (such as RT-PCR usingcomplementary primers specific for 158P3D2, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like). Alternatively, an assay for identifying a cell that expressesa 158P3D2 gene comprises detecting the presence of 158P3D2-relatedprotein in the cell or secreted by the cell. Various methods for thedetection of proteins are well known in the art and are employed for thedetection of 158P3D2-related proteins and cells that express158P3D2-related proteins. 158P3D2 expression analysis is also useful asa tool for identifying and evaluating agents that modulate 158P3D2 geneexpression. For example, 158P3D2 expression is significantly upregulatedin prostate cancer, and is expressed in cancers of the tissues listed inTable I. Identification of a molecule or biological agent that inhibits158P3D2 expression or over-expression in cancer cells is of therapeuticvalue. For example, such an agent can be identified by using a screenthat quantifies 158P3D2 expression by RT-PCR, nucleic acid hybridizationor antibody binding.

VIII.) Methods for Monitoring the Status of 158P3D2-Related Genes andtheir Products

Oncogenesis is known to be a multistep process where cellular growthbecomes progressively dysregulated and cells progress from a normalphysiological state to precancerous and then cancerous states (see,e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al.,Cancer Surv. 23: 19-32 (1995)). In this context, examining a biologicalsample for evidence of dysregulated cell growth (such as aberrant158P3D2 expression in cancers) allows for early detection of suchaberrant physiology, before a pathologic state such as cancer hasprogressed to a stage that therapeutic options are more limited and orthe prognosis is worse. In such examinations, the status of 158P3D2 in abiological sample of interest can be compared, for example, to thestatus of 158P3D2 in a corresponding normal sample (e.g. a sample fromthat individual or alternatively another individual that is not affectedby a pathology). An alteration in the status of 158P3D2 in thebiological sample (as compared to the normal sample) provides evidenceof dysregulated cellular growth. In addition to using a biologicalsample that is not affected by a pathology as a normal sample, one canalso use a predetermined normative value such as a predetermined normallevel of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol.1996 Dec. 9; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare158P3D2 status in a sample.

The term “status” in this context is used according to its art acceptedmeaning and refers to the condition or state of a gene and its products.Typically, skilled artisans use a number of parameters to evaluate thecondition or state of a gene and its products. These include, but arenot limited to the location of expressed gene products (including thelocation of 158P3D2 expressing cells) as well as the level, andbiological activity of expressed gene products (such as 158P3D2 mRNA,polynucleotides and polypeptides). Typically, an alteration in thestatus of 158P3D2 comprises a change in the location of 158P3D2 and/or158P3D2 expressing cells and/or an increase in 158P3D2 mRNA and/orprotein expression.

158P3D2 status in a sample can be analyzed by a number of means wellknown in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, Western blot analysis, and tissue arrayanalysis. Typical protocols for evaluating the status of a 158P3D2 geneand gene products are found, for example in Ausubel et al. eds., 1995,Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4(Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus,the status of 158P3D2 in a biological sample is evaluated by variousmethods utilized by skilled artisans including, but not limited togenomic Southern analysis (to examine, for example perturbations in a158P3D2 gene), Northern analysis and/or PCR analysis of 158P3D2 mRNA (toexamine, for example alterations in the polynucleotide sequences orexpression levels of 158P3D2 mRNAs), and, Western and/orimmunohistochemical analysis (to examine, for example alterations inpolypeptide sequences, alterations in polypeptide localization within asample, alterations in expression levels of 158P3D2 proteins and/orassociations of 158P3D2 proteins with polypeptide binding partners).Detectable 158P3D2 polynucleotides include, for example, a 158P3D2 geneor fragment thereof, 158P3D2 mRNA, alternative splice variants, 158P3D2mRNAs, and recombinant DNA or RNA molecules containing a 158P3D2polynucleotide.

The expression profile of 158P3D2 makes it a diagnostic marker for localand/or metastasized disease, and provides information on the growth oroncogenic potential of a biological sample. In particular, the status of158P3D2 provides information useful for predicting susceptibility toparticular disease stages, progression, and/or tumor aggressiveness. Theinvention provides methods and assays for determining 158P3D2 status anddiagnosing cancers that express 158P3D2, such as cancers of the tissueslisted in Table I. For example, because 158P3D2 mRNA is so highlyexpressed in prostate and other cancers relative to normal prostatetissue, assays that evaluate the levels of 158P3D2 mRNA transcripts orproteins in a biological sample can be used to diagnose a diseaseassociated with 158P3D2 dysregulation, and can provide prognosticinformation useful in defining appropriate therapeutic options.

The expression status of 158P3D2 provides information including thepresence, stage and location of dysplastic, precancerous and cancerouscells, predicting susceptibility to various stages of disease, and/orfor gauging tumor aggressiveness. Moreover, the expression profile makesit useful as an imaging reagent for metastasized disease. Consequently,an aspect of the invention is directed to the various molecularprognostic and diagnostic methods for examining the status of 158P3D2 inbiological samples such as those from individuals suffering from, orsuspected of suffering from a pathology characterized by dysregulatedcellular growth, such as cancer.

As described above, the status of 158P3D2 in a biological sample can beexamined by a number of well-known procedures in the art. For example,the status of 158P3D2 in a biological sample taken from a specificlocation in the body can be examined by evaluating the sample for thepresence or absence of 158P3D2 expressing cells (e.g. those that express158P3D2 mRNAs or proteins). This examination can provide evidence ofdysregulated cellular growth, for example, when 158P3D2-expressing cellsare found in a biological sample that does not normally contain suchcells (such as a lymph node), because such alterations in the status of158P3D2 in a biological sample are often associated with dysregulatedcellular growth. Specifically, one indicator of dysregulated cellulargrowth is the metastases of cancer cells from an organ of origin (suchas the prostate) to a different area of the body (such as a lymph node).In this context, evidence of dysregulated cellular growth is importantfor example because occult lymph node metastases can be detected in asubstantial proportion of patients with prostate cancer, and suchmetastases are associated with known predictors of disease progression(see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000);Su et al.,Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995August 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 158P3D2gene products by determining the status of 158P3D2 gene productsexpressed by cells from an individual suspected of having a diseaseassociated with dysregulated cell growth (such as hyperplasia or cancer)and then comparing the status so determined to the status of 158P3D2gene products in a corresponding normal sample. The presence of aberrant158P3D2 gene products in the test sample relative to the normal sampleprovides an indication of the presence of dysregulated cell growthwithin the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in 158P3D2 mRNA or protein expression in a testcell or tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of 158P3D2 mRNA can, for example, beevaluated in tissues including but not limited to those listed in TableI. The presence of significant 158P3D2 expression in any of thesetissues is useful to indicate the emergence, presence and/or severity ofa cancer, since the corresponding normal tissues do not express 158P3D2mRNA or express it at lower levels.

In a related embodiment, 158P3D2 status is determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodcomprises determining the level of 158P3D2 protein expressed by cells ina test tissue sample and comparing the level so determined to the levelof 158P3D2 expressed in a corresponding normal sample. In oneembodiment, the presence of 158P3D2 protein is evaluated, for example,using immunohistochemical methods. 158P3D2 antibodies or bindingpartners capable of detecting 158P3D2 protein expression are used in avariety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status of 158P3D2nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules. Theseperturbations can include insertions, deletions, substitutions and thelike. Such evaluations are useful because perturbations in thenucleotide and amino acid sequences are observed in a large number ofproteins associated with a growth dysregulated phenotype (see, e.g.,Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, amutation in the sequence of 158P3D2 may be indicative of the presence orpromotion of a tumor. Such assays therefore have diagnostic andpredictive value where a mutation in 158P3D2 indicates a potential lossof function or increase in tumor growth.

A wide variety of assays for observing perturbations in nucleotide andamino acid sequences are well known in the art. For example, the sizeand structure of nucleic acid or amino acid sequences of 158P3D2 geneproducts are observed by the Northern, Southern, Western, PCR and DNAsequencing protocols discussed herein. In addition, other methods forobserving perturbations in nucleotide and amino acid sequences such assingle strand conformation polymorphism analysis are well known in theart (see, e.g., U.S. Pat. No. 5,382,510 issued 7 Sep. 1999, and U.S.Pat. No. 5,952,170 issued 17 Jan. 1995).

Additionally, one can examine the methylation status of a 158P3D2 genein a biological sample. Aberrant demethylation and/or hypermethylationof CpG islands in gene 5′ regulatory regions frequently occurs inimmortalized and transformed cells, and can result in altered expressionof various genes. For example, promoter hypermethylation of the pi-classglutathione S-transferase (a protein expressed in normal prostate butnot expressed in >90% of prostate carcinomas) appears to permanentlysilence transcription of this gene and is the most frequently detectedgenomic alteration in prostate carcinomas (De Marzo et al., Am. J.Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration ispresent in at least 70% of cases of high-grade prostatic intraepithelialneoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prev.,1998, 7:531-536). In another example, expression of the LAGE-I tumorspecific gene (which is not expressed in normal prostate but isexpressed in 25-50% of prostate cancers) is induced by deoxy-azacytidinein lymphoblastoid cells, suggesting that tumoral expression is due todemethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). Avariety of assays for examining methylation status of a gene are wellknown in the art. For example, one can utilize, in Southernhybridization approaches, methylation-sensitive restriction enzymes thatcannot cleave sequences that contain methylated CpG sites to assess themethylation status of CpG islands. In addition, MSP (methylationspecific PCR) can rapidly profile the methylation status of all the CpGsites present in a CpG island of a given gene. This procedure involvesinitial modification of DNA by sodium bisulfite (which will convert allunmethylated cytosines to uracil) followed by amplification usingprimers specific for methylated versus unmethylated DNA. Protocolsinvolving methylation interference can also be found for example inCurrent Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel etal. eds., 1995.

Gene amplification is an additional method for assessing the status of158P3D2. Gene amplification is measured in a sample directly, forexample, by conventional Southern blotting or Northern blotting toquantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad.Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies are employed thatrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turnare labeled and the assay carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for thepresence of cancer cells using for example, Northern, dot blot or RT-PCRanalysis to detect 158P3D2 expression. The presence of RT-PCRamplifiable 158P3D2 mRNA provides an indication of the presence ofcancer. RT-PCR assays are well known in the art. RT-PCR detection assaysfor tumor cells in peripheral blood are currently being evaluated foruse in the diagnosis and management of a number of human solid tumors.In the prostate cancer field, these include RT-PCR assays for thedetection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol.Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000;Heston et al., 1995, Clin. Chem. 41:1687-1688).

A further aspect of the invention is an assessment of the susceptibilitythat an individual has for developing cancer. In one embodiment, amethod for predicting susceptibility to cancer comprises detecting158P3D2 mRNA or 158P3D2 protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 158P3D2 mRNAexpression correlates to the degree of susceptibility. In a specificembodiment, the presence of 158P3D2 in prostate or other tissue isexamined, with the presence of 158P3D2 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). Similarly, one can evaluate theintegrity 158P3D2 nucleotide and amino acid sequences in a biologicalsample, in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like. Thepresence of one or more perturbations in 158P3D2 gene products in thesample is an indication of cancer susceptibility (or the emergence orexistence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness.In one embodiment, a method for gauging aggressiveness of a tumorcomprises determining the level of 158P3D2 mRNA or 158P3D2 proteinexpressed by tumor cells, comparing the level so determined to the levelof 158P3D2 mRNA or 158P3D2 protein expressed in a corresponding normaltissue taken from the same individual or a normal tissue referencesample, wherein the degree of 158P3D2 mRNA or 158P3D2 protein expressionin the tumor sample relative to the normal sample indicates the degreeof aggressiveness. In a specific embodiment, aggressiveness of a tumoris evaluated by determining the extent to which 158P3D2 is expressed inthe tumor cells, with higher expression levels indicating moreaggressive tumors. Another embodiment is the evaluation of the integrityof 158P3D2 nucleotide and amino acid sequences in a biological sample,in order to identify perturbations in the structure of these moleculessuch as insertions, deletions, substitutions and the like. The presenceof one or more perturbations indicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observingthe progression of a malignancy in an individual over time. In oneembodiment, methods for observing the progression of a malignancy in anindividual over time comprise determining the level of 158P3D2 mRNA or158P3D2 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 158P3D2 mRNA or 158P3D2 proteinexpressed in an equivalent tissue sample taken from the same individualat a different time, wherein the degree of 158P3D2 mRNA or 158P3D2protein expression in the tumor sample over time provides information onthe progression of the cancer. In a specific embodiment, the progressionof a cancer is evaluated by determining 158P3D2 expression in the tumorcells over time, where increased expression over time indicates aprogression of the cancer. Also, one can evaluate the integrity 158P3D2nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like, where the presence ofone or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention is directed to methods forobserving a coincidence between the expression of 158P3D2 gene and158P3D2 gene products (or perturbations in 158P3D2 gene and 158P3D2 geneproducts) and a factor that is associated with malignancy, as a meansfor diagnosing and prognosticating the status of a tissue sample. A widevariety of factors associated with malignancy can be utilized, such asthe expression of genes associated with malignancy (e.g. PSA, PSCA andPSM expression for prostate cancer etc.) as well as gross cytologicalobservations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol.6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al.,1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg.Pathol. 23(8):918-24). Methods for observing a coincidence between theexpression of 158P3D2 gene and 158P3D2 gene products (or perturbationsin 158P3D2 gene and 158P3D2 gene products) and another factor that isassociated with malignancy are useful, for example, because the presenceof a set of specific factors that coincide with disease providesinformation crucial for diagnosing and prognosticating the status of atissue sample.

In one embodiment, methods for observing a coincidence between theexpression of 158P3D2 gene and 158P3D2 gene products (or perturbationsin 158P3D2 gene and 158P3D2 gene products) and another factor associatedwith malignancy entails detecting the overexpression of 158P3D2 mRNA orprotein in a tissue sample, detecting the overexpression of PSA mRNA orprotein in a tissue sample (or PSCA or PSM expression), and observing acoincidence of 158P3D2 mRNA or protein and PSA mRNA or proteinoverexpression (or PSCA or PSM expression). In a specific embodiment,the expression of 158P3D2 and PSA mRNA in prostate tissue is examined,where the coincidence of 158P3D2 and PSA mRNA overexpression in thesample indicates the existence of prostate cancer, prostate cancersusceptibility or the emergence or status of a prostate tumor.

Methods for detecting and quantifying the expression of 158P3D2 mRNA orprotein are described herein, and standard nucleic acid and proteindetection and quantification technologies are well known in the art.Standard methods for the detection and quantification of 158P3D2 mRNAinclude in situ hybridization using labeled 158P3D2 riboprobes, Northernblot and related techniques using 158P3D2 polynucleotide probes, RT-PCRanalysis using primers specific for 158P3D2, and other amplificationtype detection methods, such as, for example, branched DNA, SISBA, TMAand the like. In a specific embodiment, semi-quantitative RT-PCR is usedto detect and quantify 158P3D2 mRNA expression. Any number of primerscapable of amplifying 158P3D2 can be used for this purpose, includingbut not limited to the various primer sets specifically describedherein. In a specific embodiment, polyclonal or monoclonal antibodiesspecifically reactive with the wild-type 158P3D2 protein can be used inan immunohistochemical assay of biopsied tissue.

IX.) Identification of Molecules that Interact with 158P3D2

The 158P3D2 protein and nucleic acid sequences disclosed herein allow askilled artisan to identify proteins, small molecules and other agentsthat interact with 158P3D2, as well as pathways activated by 158P3D2 viaany one of a variety of art accepted protocols. For example, one canutilize one of the so-called interaction trap systems (also referred toas the “two-hybrid assay”). In such systems, molecules interact andreconstitute a transcription factor which directs expression of areporter gene, whereupon the expression of the reporter gene is assayed.Other systems identify protein-protein interactions in vivo throughreconstitution of a eukaryotic transcriptional activator, see, e.g.,U.S. Pat. No. 5,955,280 issued 21 Sep. 1999, U.S. Pat. No. 5,925,523issued 20 Jul. 1999, U.S. Pat. No. 5,846,722 issued 8 Dec. 1998 and U.S.Pat. No. 6,004,746 issued 21 Dec. 1999. Algorithms are also available inthe art for genome-based predictions of protein function (see, e.g.,Marcotte, et al., Nature 402: 4 November 1999, 83-86).

Alternatively one can screen peptide libraries to identify moleculesthat interact with 158P3D2 protein sequences. In such methods, peptidesthat bind to 158P3D2 are identified by screening libraries that encode arandom or controlled collection of amino acids. Peptides encoded by thelibraries are expressed as fusion proteins of bacteriophage coatproteins, the bacteriophage particles are then screened against the158P3D2 protein(s).

Accordingly, peptides having a wide variety of uses, such astherapeutic, prognostic or diagnostic reagents, are thus identifiedwithout any prior information on the structure of the expected ligand orreceptor molecule. Typical peptide libraries and screening methods thatcan be used to identify molecules that interact with 158P3D2 proteinsequences are disclosed for example in U.S. Pat. No. 5,723,286 issued 3Mar. 1998 and U.S. Pat. No. 5,733,731 issued 31 Mar. 1998.

Alternatively, cell lines that express 158P3D2 are used to identifyprotein-protein interactions mediated by 158P3D2. Such interactions canbe examined using immunoprecipitation techniques (see, e.g., Hamilton B.J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 158P3D2protein can be immunoprecipitated from 158P3D2-expressing cell linesusing anti-158P3D2 antibodies. Alternatively, antibodies against His-tagcan be used in a cell line engineered to express fusions of 158P3D2 anda His-tag (vectors mentioned above). The immunoprecipitated complex canbe examined for protein association by procedures such as Westernblotting, 35S-methionine labeling of proteins, protein microsequencing,silver staining and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with 158P3D2 can be identifiedthrough related embodiments of such screening assays. For example, smallmolecules can be identified that interfere with protein function,including molecules that interfere with 158P3D2's ability to mediatephosphorylation and de-phosphorylation, interaction with DNA or RNAmolecules as an indication of regulation of cell cycles, secondmessenger signaling or tumorigenesis. Similarly, small molecules thatmodulate 158P3D2-related ion channel, protein pump, or cellcommunication functions are identified and used to treat patients thathave a cancer that expresses 158P3D2 (see, e.g., Hille, B., IonicChannels of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland,Mass., 1992). Moreover, ligands that regulate 158P3D2 function can beidentified based on their ability to bind 158P3D2 and activate areporter construct. Typical methods are discussed for example in U.S.Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forminghybrid ligands in which at least one ligand is a small molecule. In anillustrative embodiment, cells engineered to express a fusion protein of158P3D2 and a DNA-binding protein are used to co-express a fusionprotein of a hybrid ligand/small molecule and a cDNA librarytranscriptional activator protein. The cells further contain a reportergene, the expression of which is conditioned on the proximity of thefirst and second fusion proteins to each other, an event that occursonly if the hybrid ligand binds to target sites on both hybrid proteins.Those cells that express the reporter gene are selected and the unknownsmall molecule or the unknown ligand is identified. This method providesa means of identifying modulators, which activate or inhibit 158P3D2.

An embodiment of this invention comprises a method of screening for amolecule that interacts with a 158P3 D2 amino acid sequence shown inFIG. 2 or FIG. 3, comprising the steps of contacting a population ofmolecules with a 158P3D2 amino acid sequence, allowing the population ofmolecules and the 158P3D2 amino acid sequence to interact underconditions that facilitate an interaction, determining the presence of amolecule that interacts with the 158P3D2 amino acid sequence, and thenseparating molecules that do not interact with the 158P3D2 amino acidsequence from molecules that do. In a specific embodiment, the methodfurther comprises purifying, characterizing and identifying a moleculethat interacts with the 158P3D2 amino acid sequence. The identifiedmolecule can be used to modulate a function performed by 158P3D2. In apreferred embodiment, the 158P3D2 amino acid sequence is contacted witha library of peptides.

X.) Therapeutic Methods and Compositions

The identification of 158P3D2 as a protein that is normally expressed ina restricted set of tissues, but which is also expressed in cancers suchas those listed in Table I, opens a number of therapeutic approaches tothe treatment of such cancers.

Of note, targeted antitumor therapies have been useful even when thetargeted protein is expressed on normal tissues, even vital normal organtissues. A vital organ is one that is necessary to sustain life, such asthe heart or colon. A non-vital organ is one that can be removedwhereupon the individual is still able to survive. Examples of non-vitalorgans are ovary, breast, and prostate.

For example, Herceptin® is an FDA approved pharmaceutical that has asits active ingredient an antibody which is immunoreactive with theprotein variously known as HER2, HER2/neu, and erb-b-2. It is marketedby Genentech and has been a commercially successful antitumor agent.Herceptin sales reached almost $400 million in 2002. Herceptin is atreatment for HER2 positive metastatic breast cancer. However, theexpression of HER2 is not limited to such tumors. The same protein isexpressed in a number of normal tissues. In particular, it is known thatHER2/neu is present in normal kidney and heart, thus these tissues arepresent in all human recipients of Herceptin. The presence of HER2/neuin normal kidney is also confirmed by Latif, Z., et al., B.J.U.International (2002) 89:5-9. As shown in this article (which evaluatedwhether renal cell carcinoma should be a preferred indication foranti-HER2 antibodies such as Herceptin) both protein and mRNA areproduced in benign renal tissues. Notably, HER2/neu protein was stronglyoverexpressed in benign renal tissue.

Despite the fact that HER2/neu is expressed in such vital tissues asheart and kidney, Herceptin is a very useful, FDA approved, andcommercially successful drug. The effect of Herceptin on cardiac tissue,i.e., “cardiotoxicity,” has merely been a side effect to treatment. Whenpatients were treated with Herceptin alone, significant cardiotoxicityoccurred in a very low percentage of patients.

Of particular note, although kidney tissue is indicated to exhibitnormal expression, possibly even higher expression than cardiac tissue,kidney has no appreciable Herceptin side effect whatsoever. Moreover, ofthe diverse array of normal tissues in which HER2 is expressed, there isvery little occurrence of any side effect. Only cardiac tissue hasmanifested any appreciable side effect at all. A tissue such as kidney,where HER2/neu expression is especially notable, has not been the basisfor any side effect.

Furthermore, favorable therapeutic effects have been found for antitumortherapies that target epidermal growth factor receptor (EGFR). EGFR isalso expressed in numerous normal tissues. There have been very limitedside effects in normal tissues following use of anti-EGFR therapeutics.

Thus, expression of a target protein in normal tissue, even vital normaltissue, does not defeat the utility of a targeting agent for the proteinas a therapeutic for certain tumors in which the protein is alsooverexpressed.

Accordingly, therapeutic approaches that inhibit the activity of a158P3D2 protein are useful for patients suffering from a cancer thatexpresses 158P3D2. These therapeutic approaches generally fall into twoclasses. One class comprises various methods for inhibiting the bindingor association of a 158P3D2 protein with its binding partner or withother proteins. Another class comprises a variety of methods forinhibiting the transcription of a 158P3D2 gene or translation of 158P3D2mRNA.

X.A.) Anti-Cancer Vaccines

The invention provides cancer vaccines comprising a 158P3D2-relatedprotein or 158P3D2-related nucleic acid. In view of the expression of158P3D2, cancer vaccines prevent and/or treat 158P3D2-expressing cancerswith minimal or no effects on non-target tissues. The use of a tumorantigen in a vaccine that generates humoral and/or cell-mediated immuneresponses as anti-cancer therapy is well known in the art and has beenemployed in prostate cancer using human PSMA and rodent PAP immunogens(Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J.Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a 158P3D2-relatedprotein, or a 158P3D2-encoding nucleic acid molecule and recombinantvectors capable of expressing and presenting the 158P3D2 immunogen(which typically comprises a number of antibody or T cell epitopes).Skilled artisans understand that a wide variety of vaccine systems fordelivery of immunoreactive epitopes are known in the art (see, e.g.,Heryln et al., Ann Med 1999 February 31(1):66-78; Maruyama et al.,Cancer Immunol Immunother 2000 June 49(3): 123-32) Briefly, such methodsof generating an immune response (e.g. humoral and/or cell-mediated) ina mammal, comprise the steps of: exposing the mammal's immune system toan immunoreactive epitope (e.g. an epitope present in a 158P3D2 proteinshown in FIG. 3 or analog or homolog thereof) so that the mammalgenerates an immune response that is specific for that epitope (e.g.generates antibodies that specifically recognize that epitope). In apreferred method, a 158P3D2 immunogen contains a biological motif, seee.g., Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from158P3D2 indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.

The entire 158P3D2 protein, immunogenic regions or epitopes thereof canbe combined and delivered by various means. Such vaccine compositionscan include, for example, lipopeptides (e.g., Vitiello, A. et al., J.Clin. Invest. 95:341, 1995), peptide compositions encapsulated inpoly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge,et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptidecompositions contained in immune stimulating complexes (ISCOMS) (see,e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin ExpImmunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs)(see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988;Tam, J.P., J. Immunol. Methods 196:17-32, 1996), peptides formulated asmultivalent peptides; peptides for use in ballistic delivery systems,typically crystallized peptides, viral delivery vectors (Perkus, M. E.et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p.379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. etal., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda,P. K. et al., Virology 175:535, 1990), particles of viral or syntheticorigin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996;Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. etal., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R.,and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al.,Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol.148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked orparticle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993;Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993;Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S.H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev.Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16,1993). Toxin-targeted delivery technologies, also known as receptormediated targeting, such as those of Avant Immunotherapeutics, Inc.(Needham, Mass.) may also be used.

In patients with 158P3D2-associated cancer, the vaccine compositions ofthe invention can also be used in conjunction with other treatments usedfor cancer, e.g., surgery, chemotherapy, drug therapies, radiationtherapies, etc. including use in combination with immune adjuvants suchas IL-2, IL-12, GM-CSF, and the like.

X.A.1. Cellular Vaccines

CTL epitopes can be determined using specific algorithms to identifypeptides within 158P3D2 protein that bind corresponding HLA alleles (seee.g., Table IV; Epimer™ and Epimatrix™, Brown University (URLbrown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and, BIMAS,(URL bimas.dcrt.nih.gov/; SYFPEITHI at URLsyfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a 158P3D2immunogen contains one or more amino acid sequences identified usingtechniques well known in the art, such as the sequences shown in TablesVIII-XXI and XXII-XLIX or a peptide of 8, 9, 10 or 11 amino acidsspecified by an HLA Class I motif/supermotif (e.g., Table IV (A), TableIV (D), or Table IV (E)) and/or a peptide of at least 9 amino acids thatcomprises an HLA Class II motif/supermotif (e.g., Table IV (B) or TableIV (C)). As is appreciated in the art, the HLA Class I binding groove isessentially closed ended so that peptides of only a particular sizerange can fit into the groove and be bound, generally HLA Class Iepitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLAClass II binding groove is essentially open ended; therefore a peptideof about 9 or more amino acids can be bound by an HLA Class II molecule.Due to the binding groove differences between HLA Class I and II, HLAClass I motifs are length specific, i.e., position two of a Class Imotif is the second amino acid in an amino to carboxyl direction of thepeptide. The amino acid positions in a Class II motif are relative onlyto each other, not the overall peptide, i.e., additional amino acids canbe attached to the amino and/or carboxyl termini of a motif-bearingsequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer than25 amino acids.

X.A.2. Antibody-Based Vaccines

A wide variety of methods for generating an immune response in a mammalare known in the art (for example as the first step in the generation ofhybridomas). Methods of generating an immune response in a mammalcomprise exposing the mammal's immune system to an immunogenic epitopeon a protein (e.g. a 158P3D2 protein) so that an immune response isgenerated. A typical embodiment consists of a method for generating animmune response to 158P3D2 in a host, by contacting the host with asufficient amount of at least one 158P3D2 B cell or cytotoxic T-cellepitope or analog thereof; and at least one periodic interval thereafterre-contacting the host with the 158P3D2 B cell or cytotoxic T-cellepitope or analog thereof. A specific embodiment consists of a method ofgenerating an immune response against a 158P3D2-related protein or aman-made multiepitopic peptide comprising: administering 158P3D2immunogen (e.g. a 158P3D2 protein or a peptide fragment thereof, a158P3D2 fusion protein or analog etc.) in a vaccine preparation to ahuman or another mammal. Typically, such vaccine preparations furthercontain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or auniversal helper epitope such as a PADRE™ peptide (Epimmune Inc., SanDiego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3);164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 andAlexander et al., Immunol. Res. 1998 18(2): 79-92). An alternativemethod comprises generating an immune response in an individual againsta 158P3D2 immunogen by: administering in vivo to muscle or skin of theindividual's body a DNA molecule that comprises a DNA sequence thatencodes a 158P3D2 immunogen, the DNA sequence operatively linked toregulatory sequences which control the expression of the DNA sequence;wherein the DNA molecule is taken up by cells, the DNA sequence isexpressed in the cells and an immune response is generated against theimmunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a geneticvaccine facilitator such as anionic lipids; saponins; lectins;estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; andurea is also administered. In addition, an antiidiotypic antibody can beadministered that mimics 158P3D2, in order to generate a response to thetarget antigen.

X.A.3. Nucleic Acid Vaccines:

Vaccine compositions of the invention include nucleic acid-mediatedmodalities.

DNA or RNA that encode protein(s) of the invention can be administeredto a patient. Genetic immunization methods can be employed to generateprophylactic or therapeutic humoral and cellular immune responsesdirected against cancer cells expressing 158P3D2. Constructs comprisingDNA encoding a 158P3D2-related protein/immunogen and appropriateregulatory sequences can be injected directly into muscle or skin of anindividual, such that the cells of the muscle or skin take-up theconstruct and express the encoded 158P3D2 protein/immunogen.Alternatively, a vaccine comprises a 158P3D2-related protein. Expressionof the 158P3D2-related protein immunogen results in the generation ofprophylactic or therapeutic humoral and cellular immunity against cellsthat bear a 158P3D2 protein. Various prophylactic and therapeuticgenetic immunization techniques known in the art can be used (forreview, see information and references published at Internet addressgenweb.com). Nucleic acid-based delivery is described, for instance, inWolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos.5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO98/04720. Examples of DNA-based delivery technologies include “nakedDNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery,cationic lipid complexes, and particle-mediated (“gene gun”) orpressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of theinvention can be expressed via viral or bacterial vectors. Various viralgene delivery systems that can be used in the practice of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol.8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)).Non-viral delivery systems can also be employed by introducing naked DNAencoding a 158P3D2-related protein into the patient (e.g.,intramuscularly or intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotidesequences that encode the peptides of the invention. Upon introductioninto a host, the recombinant vaccinia virus expresses the proteinimmunogenic peptide, and thereby elicits a host immune response.Vaccinia vectors and methods useful in immunization protocols aredescribed in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG(Bacille Calmette Guerin). BCG vectors are described in Stover et al.,Nature 351:456-460 (1991). A wide variety of other vectors useful fortherapeutic administration or immunization of the peptides of theinvention, e.g. adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, will be apparent to those skilled in the art from thedescription herein.

Thus, gene delivery systems are used to deliver a 158P3D2-relatednucleic acid molecule. In one embodiment, the full-length human 158P3D2cDNA is employed. In another embodiment, 158P3D2 nucleic acid moleculesencoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopesare employed.

X.A.4. Ex Vivo Vaccines

Various ex vivo strategies can also be employed to generate an immuneresponse. One approach involves the use of antigen presenting cells(APCs) such as dendritic cells (DC) to present 158P3D2 antigen to apatient's immune system. Dendritic cells express MHC class I and IImolecules, B7 co-stimulator, and IL-12, and are thus highly specializedantigen presenting cells. In prostate cancer, autologous dendritic cellspulsed with peptides of the prostate-specific membrane antigen (PSMA)are being used in a Phase I clinical trial to stimulate prostate cancerpatients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphyet al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used topresent 158P3D2 peptides to T cells in the context of MHC class I or IImolecules. In one embodiment, autologous dendritic cells are pulsed with158P3D2 peptides capable of binding to MHC class I and/or class IImolecules. In another embodiment, dendritic cells are pulsed with thecomplete 158P3D2 protein. Yet another embodiment involves engineeringthe overexpression of a 158P3D2 gene in dendritic cells using variousimplementing vectors known in the art, such as adenovirus (Arthur etal., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al.,1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), ortumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.186:1177-1182). Cells that express 158P3D2 can also be engineered toexpress immune modulators, such as GM-CSF, and used as immunizingagents.

X.B.) 158P3D2 as a Target for Antibody-Based Therapy

158P3D2 is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because 158P3D2 is expressed by cancer cells of various lineagesrelative to corresponding normal cells, systemic administration of158P3D2-immunoreactive compositions are prepared that exhibit excellentsensitivity without toxic, non-specific and/or non-target effects causedby binding of the immunoreactive composition to non-target organs andtissues. Antibodies specifically reactive with domains of 158P3D2 areuseful to treat 158P3D2-expressing cancers systemically, either asconjugates with a toxin or therapeutic agent, or as naked antibodiescapable of inhibiting cell proliferation or function.

158P3D2 antibodies can be introduced into a patient such that theantibody binds to 158P3D2 and modulates a function, such as aninteraction with a binding partner, and consequently mediatesdestruction of the tumor cells and/or inhibits the growth of the tumorcells. Mechanisms by which such antibodies exert a therapeutic effectcan include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulation of the physiological function of 158P3D2,inhibition of ligand binding or signal transduction pathways, modulationof tumor cell differentiation, alteration of tumor angiogenesis factorprofiles, and/or apoptosis.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of a 158P3D2 sequence shown in FIG. 2 or FIG. 3. Inaddition, skilled artisans understand that it is routine to conjugateantibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:113678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents aredelivered directly to cells, such as by conjugating them to antibodiesspecific for a molecule expressed by that cell (e.g. 158P3D2), thecytotoxic agent will exert its known biological effect (i.e.cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxicagent conjugates to kill cells are known in the art. In the context ofcancers, typical methods entail administering to an animal having atumor a biologically effective amount of a conjugate comprising aselected cytotoxic and/or therapeutic agent linked to a targeting agent(e.g. an anti-158P3D2 antibody) that binds to a marker (e.g. 158P3D2)expressed, accessible to binding or localized on the cell surfaces. Atypical embodiment is a method of delivering a cytotoxic and/ortherapeutic agent to a cell expressing 158P3D2, comprising conjugatingthe cytotoxic agent to an antibody that immunospecifically binds to a158P3D2 epitope, and, exposing the cell to the antibody-agent conjugate.Another illustrative embodiment is a method of treating an individualsuspected of suffering from metastasized cancer, comprising a step ofadministering parenterally to said individual a pharmaceuticalcomposition comprising a therapeutically effective amount of an antibodyconjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-158P3D2 antibodies can be done inaccordance with various approaches that have been successfully employedin the treatment of other types of cancer, including but not limited tocolon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138),multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari etal., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992,Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al.,1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994,Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin or radioisotope, such as the conjugation ofY91 or I131 to anti-CD20 antibodies (e.g., Zevalin™, IDECPharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals), while othersinvolve co-administration of antibodies and other therapeutic agents,such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). Theantibodies can be conjugated to a therapeutic agent. To treat prostatecancer, for example, 158P3D2 antibodies can be administered inconjunction with radiation, chemotherapy or hormone ablation. Also,antibodies can be conjugated to a toxin such as calicheamicin (e.g.,Mylotarg™, Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG4kappa antibody conjugated to antitumor antibiotic calicheamicin) or amaytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform,ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).

Although 158P3D2 antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well. Fan et al. (Cancer Res.53:4637-4642, 1993), Prewett et al. (International J. of Onco.9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991)describe the use of various antibodies together with chemotherapeuticagents.

Although 158P3D2 antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 158P3D2expression, preferably using immunohistochemical assessments of tumortissue, quantitative 158P3D2 imaging, or other techniques that reliablyindicate the presence and degree of 158P3D2 expression.Immunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

Anti-158P3D2 monoclonal antibodies that treat prostate and other cancersinclude those that initiate a potent immune response against the tumoror those that are directly cytotoxic. In this regard, anti-158P3D2monoclonal antibodies (mAbs) can elicit tumor cell lysis by eithercomplement-mediated or antibody-dependent cell cytotoxicity (ADCC)mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites on complement proteins. In addition, anti-158P3D2 mAbs that exerta direct biological effect on tumor growth are useful to treat cancersthat express 158P3D2. Mechanisms by which directly cytotoxic mAbs actinclude: inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism(s) by which a particularanti-158P3D2 mAb exerts an anti-tumor effect is evaluated using anynumber of in vitro assays that evaluate cell death such as ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

In some patients, the use of murine or other non-human monoclonalantibodies, or human/mouse chimeric mAbs can induce moderate to strongimmune responses against the non-human antibody. This can result inclearance of the antibody from circulation and reduced efficacy. In themost severe cases, such an immune response can lead to the extensiveformation of immune complexes which, potentially, can cause renalfailure. Accordingly, preferred monoclonal antibodies used in thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 158P3D2antigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-158P3D2 mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails can have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination canexhibit synergistic therapeutic effects. In addition, anti-158P3D2 mAbscan be administered concomitantly with other therapeutic modalities,including but not limited to various chemotherapeutic agents,androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery orradiation. The anti-158P3D2 mAbs are administered in their “naked” orunconjugated form, or can have a therapeutic agent(s) conjugated tothem.

Anti-158P3D2 antibody formulations are administered via any routecapable of delivering the antibodies to a tumor cell. Routes ofadministration include, but are not limited to, intravenous,intraperitoneal, intramuscular, intratumor, intradermal, and the like.Treatment generally involves repeated administration of the anti-158P3D2antibody preparation, via an acceptable route of administration such asintravenous injection (IV), typically at a dose in the range of about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of10-1000 mg mAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin™ mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kgIV of the anti-158P3D2 mAb preparation represents an acceptable dosingregimen. Preferably, the initial loading dose is administered as a90-minute or longer infusion. The periodic maintenance dose isadministered as a 30 minute or longer infusion, provided the initialdose was well tolerated. As appreciated by those of skill in the art,various factors can influence the ideal dose regimen in a particularcase. Such factors include, for example, the binding affinity and halflife of the Ab or mAbs used, the degree of 158P3D2 expression in thepatient, the extent of circulating shed 158P3D2 antigen, the desiredsteady-state antibody concentration level, frequency of treatment, andthe influence of chemotherapeutic or other agents used in combinationwith the treatment method of the invention, as well as the health statusof a particular patient.

Optionally, patients should be evaluated for the levels of 158P3D2 in agiven sample (e.g. the levels of circulating 158P3D2 antigen and/or158P3D2 expressing cells) in order to assist in the determination of themost effective dosing regimen, etc. Such evaluations are also used formonitoring purposes throughout therapy, and are useful to gaugetherapeutic success in combination with the evaluation of otherparameters (for example, urine cytology and/or ImmunoCyt levels inbladder cancer therapy, or by analogy, serum PSA levels in prostatecancer therapy).

Anti-idiotypic anti-158P3D2 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga 158P3D2-related protein. In particular, the generation ofanti-idiotypic antibodies is well known in the art; this methodology canreadily be adapted to generate anti-idiotypic anti-158P3D2 antibodiesthat mimic an epitope on a 158P3D2-related protein (see, for example,Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin.Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother.43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccinestrategies.

X.C.) 158P3D2 as a Target for Cellular Immune Responses

Vaccines and methods of preparing vaccines that contain animmunogenically effective amount of one or more HLA-binding peptides asdescribed herein are further embodiments of the invention. Furthermore,vaccines in accordance with the invention encompass compositions of oneor more of the claimed peptides. A peptide can be present in a vaccineindividually. Alternatively, the peptide can exist as a homopolymercomprising multiple copies of the same peptide, or as a heteropolymer ofvarious peptides. Polymers have the advantage of increased immunologicalreaction and, where different peptide epitopes are used to make up thepolymer, the additional ability to induce antibodies and/or CTLs thatreact with different antigenic determinants of the pathogenic organismor tumor-related peptide targeted for an immune response. Thecomposition can be a naturally occurring region of an antigen or can beprepared, e.g., recombinantly or by chemical synthesis.

Carriers that can be used with vaccines of the invention are well knownin the art, and include, e.g., thyroglobulin, albumins such as humanserum albumin, tetanus toxoid, polyamino acids such as poly 1-lysine,poly 1-glutamic acid, influenza, hepatitis B virus core protein, and thelike. The vaccines can contain a physiologically tolerable (i.e.,acceptable) diluent such as water, or saline, preferably phosphatebuffered saline. The vaccines also typically include an adjuvant.Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate,aluminum hydroxide, or alum are examples of materials well known in theart. Additionally, as disclosed herein, CTL responses can be primed byconjugating peptides of the invention to lipids, such astripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS). Moreover, anadjuvant such as a syntheticcytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotideshas been found to increase CTL responses 10- to 100-fold. (see, e.g.Davila and Celis, J. Immunol. 165:539-547 (2000)).

Upon immunization with a peptide composition in accordance with theinvention, via injection, aerosol, oral, transdermal, transmucosal,intrapleural, intrathecal, or other suitable routes, the immune systemof the host responds to the vaccine by producing large amounts of CTLsand/or HTLs specific for the desired antigen. Consequently, the hostbecomes at least partially immune to later development of cells thatexpress or overexpress 158P3D2 antigen, or derives at least sometherapeutic benefit when the antigen was tumor-associated.

In some embodiments, it may be desirable to combine the class I peptidecomponents with components that induce or facilitate neutralizingantibody and or helper T cell responses directed to the target antigen.A preferred embodiment of such a composition comprises class I and classII epitopes in accordance with the invention. An alternative embodimentof such a composition comprises a class I and/or class II epitope inaccordance with the invention, along with a cross reactive HTL epitopesuch as PADRE™ (Epimmune, San Diego, Calif.) molecule (described e.g.,in U.S. Pat. No. 5,736,142).

A vaccine of the invention can also include antigen-presenting cells(APC), such as dendritic cells (DC), as a vehicle to present peptides ofthe invention. Vaccine compositions can be created in vitro, followingdendritic cell mobilization and harvesting, whereby loading of dendriticcells occurs in vitro. For example, dendritic cells are transfected,e.g., with a minigene in accordance with the invention, or are pulsedwith peptides. The dendritic cell can then be administered to a patientto elicit immune responses in vivo. Vaccine compositions, either DNA- orpeptide-based, can also be administered in vivo in combination withdendritic cell mobilization whereby loading of dendritic cells occurs invivo.

Preferably, the following principles are utilized when selecting anarray of epitopes for inclusion in a polyepitopic composition for use ina vaccine, or for selecting discrete epitopes to be included in avaccine and/or to be encoded by nucleic acids such as a minigene. It ispreferred that each of the following principles be balanced in order tomake the selection. The multiple epitopes to be incorporated in a givenvaccine composition may be, but need not be, contiguous in sequence inthe native antigen from which the epitopes are derived.

-   -   1.) Epitopes are selected which, upon administration, mimic        immune responses that have been observed to be correlated with        tumor clearance. For HLA Class I this includes 3-4 epitopes that        come from at least one tumor associated antigen (TAA). For HLA        Class II a similar rationale is employed; again 3-4 epitopes are        selected from at least one TAA (see, e.g., Rosenberg et al.,        Science 278:1447-1450). Epitopes from one TAA may be used in        combination with epitopes from one or more additional TAAs to        produce a vaccine that targets tumors with varying expression        patterns of frequently-expressed TAAs.    -   2.) Epitopes are selected that have the requisite binding        affinity established to be correlated with immunogenicity: for        HLA Class I an IC50 of 500 nM or less, often 200 nM or less; and        for Class II an IC50 of 1000 nM or less.    -   3.) Sufficient supermotif bearing-peptides, or a sufficient        array of allele-specific motif-bearing peptides, are selected to        give broad population coverage. For example, it is preferable to        have at least 80% population coverage. A Monte Carlo analysis, a        statistical evaluation known in the art, can be employed to        assess the breadth, or redundancy of, population coverage.    -   4.) When selecting epitopes from cancer-related antigens it is        often useful to select analogs because the patient may have        developed tolerance to the native epitope.    -   5.) Of particular relevance are epitopes referred to as “nested        epitopes.” Nested epitopes occur where at least two epitopes        overlap in a given peptide sequence. A nested peptide sequence        can comprise B cell, HLA class I and/or HLA class II epitopes.        When providing nested epitopes, a general objective is to        provide the greatest number of epitopes per sequence. Thus, an        aspect is to avoid providing a peptide that is any longer than        the amino terminus of the amino terminal epitope and the        carboxyl terminus of the carboxyl terminal epitope in the        peptide. When providing a multi-epitopic sequence, such as a        sequence comprising nested epitopes, it is generally important        to screen the sequence in order to insure that it does not have        pathological or other deleterious biological properties.    -   6.) If a polyepitopic protein is created, or when creating a        minigene, an objective is to generate the smallest peptide that        encompasses the epitopes of interest. This principle is similar,        if not the same as that employed when selecting a peptide        comprising nested epitopes. However, with an artificial        polyepitopic peptide, the size minimization objective is        balanced against the need to integrate any spacer sequences        between epitopes in the polyepitopic protein. Spacer amino acid        residues can, for example, be introduced to avoid junctional        epitopes (an epitope recognized by the immune system, not        present in the target antigen, and only created by the man-made        juxtaposition of epitopes), or to facilitate cleavage between        epitopes and thereby enhance epitope presentation. Junctional        epitopes are generally to be avoided because the recipient may        generate an immune response to that non-native epitope. Of        particular concern is a junctional epitope that is a “dominant        epitope.” A dominant epitope may lead to such a zealous response        that immune responses to other epitopes are diminished or        suppressed.    -   7.) Where the sequences of multiple variants of the same target        protein are present, potential peptide epitopes can also be        selected on the basis of their conservancy. For example, a        criterion for conservancy may define that the entire sequence of        an HLA class I binding peptide or the entire 9-mer core of a        class II binding peptide be conserved in a designated percentage        of the sequences evaluated for a specific protein antigen.

X.C.1. Minigene Vaccines

A number of different approaches are available which allow simultaneousdelivery of multiple epitopes. Nucleic acids encoding the peptides ofthe invention are a particularly useful embodiment of the invention.Epitopes for inclusion in a minigene are preferably selected accordingto the guidelines set forth in the previous section. A preferred meansof administering nucleic acids encoding the peptides of the inventionuses minigene constructs encoding a peptide comprising one or multipleepitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka etal., J.

Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J. Virol.71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996;Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine16:426, 1998. For example, a multi-epitope DNA plasmid encodingsupermotif- and/or motif-bearing epitopes derived 158P3D2, the PADRE®universal helper T cell epitope or multiple HTL epitopes from 158P3D2(see e.g., Tables VIII-XXI and XXII to XLIX), and an endoplasmicreticulum-translocating signal sequence can be engineered. A vaccine mayalso comprise epitopes that are derived from other TAAs.

The immunogenicity of a multi-epitopic minigene can be confirmed intransgenic mice to evaluate the magnitude of CTL induction responsesagainst the epitopes tested. Further, the immunogenicity of DNA-encodedepitopes in vivo can be correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid. Thus, these experiments can show that the minigene serves toboth: 1.) generate a CTL response and 2.) that the induced CTLsrecognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes(minigene) for expression in human cells, the amino acid sequences ofthe epitopes may be reverse translated. A human codon usage table can beused to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat can be reverse translated and included in the minigene sequenceinclude: HLA class I epitopes, HLA class II epitopes, antibody epitopes,a ubiquitination signal sequence, and/or an endoplasmic reticulumtargeting signal. In addition, HLA presentation of CTL and HTL epitopesmay be improved by including synthetic (e.g. poly-alanine) ornaturally-occurring flanking sequences adjacent to the CTL or HTLepitopes; these larger peptides comprising the epitope(s) are within thescope of the invention.

The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

Standard regulatory sequences well known to those of skill in the artare preferably included in the vector to ensure expression in the targetcells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play arole in the immunogenicity of DNA vaccines. These sequences may beincluded in the vector, outside the minigene coding sequence, if desiredto enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allowsproduction of both the minigene-encoded epitopes and a second protein(included to enhance or decrease immunogenicity) can be used. Examplesof proteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF),cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, orfor HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego,Calif.). Helper (HTL) epitopes can be joined to intracellular targetingsignals and expressed separately from expressed CTL epitopes; thisallows direction of the HTL epitopes to a cell compartment differentthan that of the CTL epitopes. If required, this could facilitate moreefficient entry of HTL epitopes into the HLA class II pathway, therebyimproving HTL induction. In contrast to HTL or CTL induction,specifically decreasing the immune response by co-expression ofimmunosuppressive molecules (e.g. TGF-β) may be beneficial in certaindiseases.

Therapeutic quantities of plasmid DNA can be produced for example, byfermentation in E. coli, followed by purification. Aliquots from theworking cell bank are used to inoculate growth medium, and grown tosaturation in shaker flasks or a bioreactor according to well-knowntechniques. Plasmid DNA can be purified using standard bioseparationtechnologies such as solid phase anion-exchange resins supplied byQIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can beisolated from the open circular and linear forms using gelelectrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety offormulations.

The simplest of these is reconstitution of lyophilized DNA in sterilephosphate-buffer saline (PBS). This approach, known as “naked DNA,” iscurrently being used for intramuscular (IM) administration in clinicaltrials. To maximize the immunotherapeutic effects of minigene DNAvaccines, an alternative method for formulating purified plasmid DNA maybe desirable. A variety of methods have been described, and newtechniques may become available. Cationic lipids, glycolipids, andfusogenic liposomes can also be used in the formulation (see, e.g., asdescribed by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7):682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al.,Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides andcompounds referred to collectively as protective, interactive,non-condensing compounds (PINC) could also be complexed to purifiedplasmid DNA to influence variables such as stability, intramusculardispersion, or trafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay forexpression and HLA class I presentation of minigene-encoded CTLepitopes. For example, the plasmid DNA is introduced into a mammaliancell line that is suitable as a target for standard CTL chromium releaseassays. The transfection method used will be dependent on the finalformulation. Electroporation can be used for “naked” DNA, whereascationic lipids allow direct in vitro transfection. A plasmid expressinggreen fluorescent protein (GFP) can be co-transfected to allowenrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (51Cr) labeled and usedas target cells for epitope-specific CTL lines; cytolysis, detected by51Cr release, indicates both production of, and HLA presentation of,minigene-encoded CTL epitopes. Expression of HTL epitopes may beevaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanHLA proteins are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g., IM for DNA in PBS,intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days afterimmunization, splenocytes are harvested and restimulated for one week inthe presence of peptides encoding each epitope being tested. Thereafter,for CTL effector cells, assays are conducted for cytolysis ofpeptide-loaded, 51Cr-labeled target cells using standard techniques.Lysis of target cells that were sensitized by HLA loaded with peptideepitopes, corresponding to minigene-encoded epitopes, demonstrates DNAvaccine function for in vivo induction of CTLs. Immunogenicity of HTLepitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253. Usingthis technique, particles comprised solely of DNA are administered. In afurther alternative embodiment, DNA can be adhered to particles, such asgold particles.

Minigenes can also be delivered using other bacterial or viral deliverysystems well known in the art, e.g., an expression construct encodingepitopes of the invention can be incorporated into a viral vector suchas vaccinia.

X.C.2. Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising CTL peptides of the invention can bemodified, e.g., analoged, to provide desired attributes, such asimproved serum half life, broadened population coverage or enhancedimmunogenicity.

For instance, the ability of a peptide to induce CTL activity can beenhanced by linking the peptide to a sequence which contains at leastone epitope that is capable of inducing a T helper cell response.Although a CTL peptide can be directly linked to a T helper peptide,often CTL epitope/HTL epitope conjugates are linked by a spacermolecule. The spacer is typically comprised of relatively small, neutralmolecules, such as amino acids or amino acid mimetics, which aresubstantially uncharged under physiological conditions. The spacers aretypically selected from, e.g., Ala, Gly, or other neutral spacers ofnonpolar amino acids or neutral polar amino acids. It will be understoodthat the optionally present spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. When present, thespacer will usually be at least one or two residues, more usually threeto six residues and sometimes 10 or more residues. The CTL peptideepitope can be linked to the T helper peptide epitope either directly orvia a spacer either at the amino or carboxy terminus of the CTL peptide.The amino terminus of either the immunogenic peptide or the T helperpeptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognizedby T helper cells present in a majority of a genetically diversepopulation. This can be accomplished by selecting peptides that bind tomany, most, or all of the HLA class II molecules. Examples of such aminoacid bind many HLA Class II molecules include sequences from antigenssuch as tetanus toxoid at positions 830-843 QYIKANSKFIGITE; (SEQ ID NO:63), Plasmodium falciparum circumsporozoite (CS) protein at positions378-398 DIEKKIAKMEKASSVFNVVNS; (SEQ ID NO: 64), and Streptococcus 18 kDprotein at positions 116-131 GAVDSILGGVATYGAA; (SEQ ID NO: 65). Otherexamples include peptides bearing a DR 1-4-7 supermotif, or either ofthe DR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable ofstimulating T helper lymphocytes, in a loosely HLA-restricted fashion,using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds calledPan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego,Calif.) are designed, most preferably, to bind most HLA-DR (human HLAclass II) molecules. For instance, a pan-DR-binding epitope peptidehaving the formula: xKXVAAWTLKAAx (SEQ ID NO: 66), where “X” is eithercyclohexylalanine, phenylalanine, or tyrosine, and a is either d-alanineor 1-alanine, has been found to bind to most HLA-DR alleles, and tostimulate the response of T helper lymphocytes from most individuals,regardless of their HLA type. An alternative of a pan-DR binding epitopecomprises all “L” natural amino acids and can be provided in the form ofnucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biologicalproperties. For example, they can be modified to include d-amino acidsto increase their resistance to proteases and thus extend their serumhalf life, or they can be conjugated to other molecules such as lipids,proteins, carbohydrates, and the like to increase their biologicalactivity. For example, a T helper peptide can be conjugated to one ormore palmitic acid chains at either the amino or carboxyl termini.

X.C.3. Combinations of CTL Peptides with T Cell Priming Agents

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primes Blymphocytes or T lymphocytes. Lipids have been identified as agentscapable of priming CTL in vivo. For example, palmitic acid residues canbe attached to the ε- and α-amino groups of a lysine residue and thenlinked, e.g., via one or more linking residues such as Gly, Gly-Gly-,Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidatedpeptide can then be administered either directly in a micelle orparticle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. In a preferred embodiment, aparticularly effective immunogenic composition comprises palmitic acidattached to ε- and α-amino groups of Lys, which is attached via linkage,e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P3CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide (see, e.g., Deres, et al., Nature 342:561,1989). Peptides of the invention can be coupled to P3CSS, for example,and the lipopeptide administered to an individual to prime specificallyan immune response to the target antigen. Moreover, because theinduction of neutralizing antibodies can also be primed withP3CSS-conjugated epitopes, two such compositions can be combined to moreeffectively elicit both humoral and cell-mediated responses.

X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTLPeptides

An embodiment of a vaccine composition in accordance with the inventioncomprises ex vivo administration of a cocktail of epitope-bearingpeptides to PBMC, or isolated DC therefrom, from the patient's blood. Apharmaceutical to facilitate harvesting of DC can be used, such asProgenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) or GM-CSF/IL-4.After pulsing the DC with peptides and prior to reinfusion intopatients, the DC are washed to remove unbound peptides. In thisembodiment, a vaccine comprises peptide-pulsed DCs which present thepulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of whichstimulate CTL responses to 158P3D2. Optionally, a helper T cell (HTL)peptide, such as a natural or artificial loosely restricted HLA Class IIpeptide, can be included to facilitate the CTL response. Thus, a vaccinein accordance with the invention is used to treat a cancer whichexpresses or overexpresses 158P3D2.

X.D.) Adoptive Immunotherapy

Antigenic 158P3D2-related peptides are used to elicit a CTL and/or HTLresponse ex vivo, as well. The resulting CTL or HTL cells, can be usedto treat tumors in patients that do not respond to other conventionalforms of therapy, or will not respond to a therapeutic vaccine peptideor nucleic acid in accordance with the invention. Ex vivo CTL or HTLresponses to a particular antigen are induced by incubating in tissueculture the patient's, or genetically compatible, CTL or HTL precursorcells together with a source of antigen-presenting cells (APC), such asdendritic cells, and the appropriate immunogenic peptide. After anappropriate incubation time (typically about 7-28 days), in which theprecursor cells are activated and expanded into effector cells, thecells are infused back into the patient, where they will destroy (CTL)or facilitate destruction (HTL) of their specific target cell (e.g., atumor cell). Transfected dendritic cells may also be used as antigenpresenting cells.

X.E.) Administration of Vaccines for Therapeutic or ProphylacticPurposes

Pharmaceutical and vaccine compositions of the invention are typicallyused to treat and/or prevent a cancer that expresses or overexpresses158P3D2. In therapeutic applications, peptide and/or nucleic acidcompositions are administered to a patient in an amount sufficient toelicit an effective B cell, CTL and/or HTL response to the antigen andto cure or at least partially arrest or slow symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the particular composition administered, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of theinvention, or DNA encoding them, are generally administered to anindividual already bearing a tumor that expresses 158P3D2. The peptidesor DNA encoding them can be administered individually or as fusions ofone or more peptide sequences. Patients can be treated with theimmunogenic peptides separately or in conjunction with other treatments,such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the firstdiagnosis of 158P3D2-associated cancer. This is followed by boostingdoses until at least symptoms are substantially abated and for a periodthereafter. The embodiment of the vaccine composition (i.e., including,but not limited to embodiments such as peptide cocktails, polyepitopicpolypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells)delivered to the patient may vary according to the stage of the diseaseor the patient's health status. For example, in a patient with a tumorthat expresses 158P3D2, a vaccine comprising 158P3D2-specific CTL may bemore efficacious in killing tumor cells in patient with advanced diseasethan alternative embodiments.

It is generally important to provide an amount of the peptide epitopedelivered by a mode of administration sufficient to stimulateeffectively a cytotoxic T cell response; compositions which stimulatehelper T cell responses can also be given in accordance with thisembodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in aunit dosage range where the lower value is about 1, 5, 50, 500, or 1,000μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg.Dosage values for a human typically range from about 500 μg to about50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0μg to about 50,000 μg of peptide pursuant to a boosting regimen overweeks to months may be administered depending upon the patient'sresponse and condition as determined by measuring the specific activityof CTL and HTL obtained from the patient's blood. Administration shouldcontinue until at least clinical symptoms or laboratory tests indicatethat the neoplasia, has been eliminated or reduced and for a periodthereafter. The dosages, routes of administration, and dose schedulesare adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the presentinvention are employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, as a result of the minimal amounts of extraneous substances andthe relative nontoxic nature of the peptides in preferred compositionsof the invention, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely asprophylactic agents. Generally the dosage for an initial prophylacticimmunization generally occurs in a unit dosage range where the lowervalue is about 1, 5, 50, 500, or 1000 μg and the higher value is about10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a humantypically range from about 500 μg to about 50,000 μg per 70 kilogrampatient. This is followed by boosting dosages of between about 1.0 μg toabout 50,000 μg of peptide administered at defined intervals from aboutfour weeks to six months after the initial administration of vaccine.The immunogenicity of the vaccine can be assessed by measuring thespecific activity of CTL and HTL obtained from a sample of the patient'sblood.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral, nasal, intrathecal, or local (e.g. as acream or topical ointment) administration. Preferably, thepharmaceutical compositions are administered parentally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the immunogenic peptides dissolved or suspendedin an acceptable carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, e.g., water, buffered water,0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH-adjusting and buffering agents, tonicity adjusting agents, wettingagents, preservatives, and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.1%, usuallyat or at least about 2% to as much as 20% to 50% or more by weight, andwill be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

A human unit dose form of a composition is typically included in apharmaceutical composition that comprises a human unit dose of anacceptable carrier, in one embodiment an aqueous carrier, and isadministered in a volume/quantity that is known by those of skill in theart to be used for administration of such compositions to humans (see,e.g., Remington's Pharmaceutical Sciences, 17th Edition, A. Gennaro,Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptidedose for initial immunization can be from about 1 to about 50,000 μg,generally 100-5,000 μg, for a 70 kg patient. For example, for nucleicacids an initial immunization may be performed using an expressionvector in the form of naked nucleic acid administered IM (or SC or ID)in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to1000 μg) can also be administered using a gene gun. Following anincubation period of 3-4 weeks, a booster dose is then administered. Thebooster can be recombinant fowlpox virus administered at a dose of 5-107to 5×109 pfu.

For antibodies, a treatment generally involves repeated administrationof the anti-158P3D2 antibody preparation, via an acceptable route ofadministration such as intravenous injection (IV), typically at a dosein the range of about 0.1 to about 10 mg/kg body weight. In general,doses in the range of 10-500 mg mAb per week are effective and welltolerated. Moreover, an initial loading dose of approximately 4 mg/kgpatient body weight IV, followed by weekly doses of about 2 mg/kg IV ofthe anti-158P3D2 mAb preparation represents an acceptable dosingregimen. As appreciated by those of skill in the art, various factorscan influence the ideal dose in a particular case. Such factors include,for example, half life of a composition, the binding affinity of an Ab,the immunogenicity of a substance, the degree of 158P3D2 expression inthe patient, the extent of circulating shed 158P3D2 antigen, the desiredsteady-state concentration level, frequency of treatment, and theinfluence of chemotherapeutic or other agents used in combination withthe treatment method of the invention, as well as the health status of aparticular patient. Non-limiting preferred human unit doses are, forexample, 500 μg-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg,800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, thedose is in a range of 2-5 mg/kg body weight, e.g., with follow on weeklydoses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg bodyweight followed, e.g., in two, three or four weeks by weekly doses;0.5-10 mg/kg body weight, e.g., followed in two, three or four weeks byweekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m2 of body areaweekly; 1-600 mg m2 of body area weekly; 225-400 mg m2 of body areaweekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7,8, 9, 19, 11, 12 or more weeks.

In one embodiment, human unit dose forms of polynucleotides comprise asuitable dosage range or effective amount that provides any therapeuticeffect. As appreciated by one of ordinary skill in the art a therapeuticeffect depends on a number of factors, including the sequence of thepolynucleotide, molecular weight of the polynucleotide and route ofadministration. Dosages are generally selected by the physician or otherhealth care professional in accordance with a variety of parametersknown in the art, such as severity of symptoms, history of the patientand the like. Generally, for a polynucleotide of about 20 bases, adosage range may be selected from, for example, an independentlyselected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to anindependently selected upper limit, greater than the lower limit, ofabout 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dosemay be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg,0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg.Generally, parenteral routes of administration may require higher dosesof polynucleotide compared to more direct application to the nucleotideto diseased tissue, as do polynucleotides of increasing length.

In one embodiment, human unit dose forms of T-cells comprise a suitabledosage range or effective amount that provides any therapeutic effect.As appreciated by one of ordinary skill in the art, a therapeutic effectdepends on a number of factors. Dosages are generally selected by thephysician or other health care professional in accordance with a varietyof parameters known in the art, such as severity of symptoms, history ofthe patient and the like. A dose may be about 104 cells to about 106cells, about 106 cells to about 108 cells, about 108 to about 1011cells, or about 108 to about 5×1010 cells. A dose may also about 106cells/m2 to about 1010 cells/m2, or about 106 cells/m2 to about 108cells/m2.

Proteins(s) of the invention, and/or nucleic acids encoding theprotein(s), can also be administered via liposomes, which may also serveto: 1) target the proteins(s) to a particular tissue, such as lymphoidtissue; 2) to target selectively to diseases cells; or, 3) to increasethe half-life of the peptide composition. Liposomes include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations, thepeptide to be delivered is incorporated as part of a liposome, alone orin conjunction with a molecule which binds to a receptor prevalent amonglymphoid cells, such as monoclonal antibodies which bind to the CD45antigen, or with other therapeutic or immunogenic compositions. Thus,liposomes either filled or decorated with a desired peptide of theinvention can be directed to the site of lymphoid cells, where theliposomes then deliver the peptide compositions. Liposomes for use inaccordance with the invention are formed from standard vesicle-forminglipids, which generally include neutral and negatively chargedphospholipids and a sterol, such as cholesterol. The selection of lipidsis generally guided by consideration of, e.g., liposome size, acidlability and stability of the liposomes in the blood stream. A varietyof methods are available for preparing liposomes, as described in, e.g.,Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat.Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporatedinto the liposome can include, e.g., antibodies or fragments thereofspecific for cell surface determinants of the desired immune systemcells. A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the peptide beingdelivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, immunogenic peptides are preferably suppliedin finely divided form along with a surfactant and propellant. Typicalpercentages of peptides are about 0.01%-20% by weight, preferably about1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from about 6 to 22 carbonatoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute about 0.1%-20%by weight of the composition, preferably about 0.25-5%. The balance ofthe composition is ordinarily propellant. A carrier can also beincluded, as desired, as with, e.g., lecithin for intranasal delivery.

XI.) Diagnostic and Prognostic Embodiments of 158P3D2.

As disclosed herein, 158P3D2 polynucleotides, polypeptides, reactivecytotoxic T cells (CTL), reactive helper T cells (HTL) andanti-polypeptide antibodies are used in well known diagnostic,prognostic and therapeutic assays that examine conditions associatedwith dysregulated cell growth such as cancer, in particular the cancerslisted in Table I (see, e.g., both its specific pattern of tissueexpression as well as its overexpression in certain cancers as describedfor example in the Example entitled “Expression analysis of 158P3D2 innormal tissues, and patient specimens”).

158P3D2 can be analogized to a prostate associated antigen PSA, thearchetypal marker that has been used by medical practitioners for yearsto identify and monitor the presence of prostate cancer (see, e.g.,Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J.Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. CancerInst. 91(19): 1635-1640(1999)). A variety of other diagnostic markersare also used in similar contexts including p53 and K-ras (see, e.g.,Tulchinsky et al., Int J Mol Med 1999 July 4(1):99-102 and Minimoto etal., Cancer Detect Prev 2000;24(1):1-12). Therefore, this disclosure of158P3D2 polynucleotides and polypeptides (as well as 158P3D2polynucleotide probes and anti-158P3D2 antibodies used to identify thepresence of these molecules) and their properties allows skilledartisans to utilize these molecules in methods that are analogous tothose used, for example, in a variety of diagnostic assays directed toexamining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 158P3D2polynucleotides, polypeptides, reactive T cells and antibodies areanalogous to those methods from well-established diagnostic assays,which employ, e.g., PSA polynucleotides, polypeptides, reactive T cellsand antibodies. For example, just as PSA polynucleotides are used asprobes (for example in Northern analysis, see, e.g., Sharief et al.,Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example inPCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190(2000)) to observe the presence and/or the level of PSA mRNAs in methodsof monitoring PSA overexpression or the metastasis of prostate cancers,the 158P3D2 polynucleotides described herein can be utilized in the sameway to detect 158P3D2 overexpression or the metastasis of prostate andother cancers expressing this gene. Alternatively, just as PSApolypeptides are used to generate antibodies specific for PSA which canthen be used to observe the presence and/or the level of PSA proteins inmethods to monitor PSA protein overexpression (see, e.g., Stephan etal., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells(see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the158P3D2 polypeptides described herein can be utilized to generateantibodies for use in detecting 158P3D2 overexpression or the metastasisof prostate cells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cellsfrom an organ of origin (such as the lung or prostate gland etc.) to adifferent area of the body (such as a lymph node), assays which examinea biological sample for the presence of cells expressing 158P3D2polynucleotides and/or polypeptides can be used to provide evidence ofmetastasis. For example, when a biological sample from tissue that doesnot normally contain 158P3D2-expressing cells (lymph node) is found tocontain 158P3D2-expressing cells such as the 158P3D2 expression seen inLAPC4 and LAPC9, xenografts isolated from lymph node and bonemetastasis, respectively, this finding is indicative of metastasis.

Alternatively 158P3D2 polynucleotides and/or polypeptides can be used toprovide evidence of cancer, for example, when cells in a biologicalsample that do not normally express 158P3D2 or express 158P3D2 at adifferent level are found to express 158P3D2 or have an increasedexpression of 158P3D2 (see, e.g., the 158P3D2 expression in the cancerslisted in Table I and in patient samples etc. shown in the accompanyingFigures). In such assays, artisans may further wish to generatesupplementary evidence of metastasis by testing the biological samplefor the presence of a second tissue restricted marker (in addition to158P3D2) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res.Pract. 192(3): 233-237 (1996)).

The use of immunohistochemistry to identify the presence of a 158P3D2polypeptide within a tissue section can indicate an altered state ofcertain cells within that tissue. It is well understood in the art thatthe ability of an antibody to localize to a polypeptide that isexpressed in cancer cells is a way of diagnosing presence of disease,disease stage, progression and/or tumor aggressiveness. Such an antibodycan also detect an altered distribution of the polypeptide within thecancer cells, as compared to corresponding non-malignant tissue.

The 158P3D2 polypeptide and immunogenic compositions are also useful inview of the phenomena of altered subcellular protein localization indisease states. Alteration of cells from normal to diseased state causeschanges in cellular morphology and is often associated with changes insubcellular protein localization/distribution. For example, cellmembrane proteins that are expressed in a polarized manner in normalcells can be altered in disease, resulting in distribution of theprotein in a non-polar manner over the whole cell surface.

The phenomenon of altered subcellular protein localization in a diseasestate has been demonstrated with MUC1 and Her2 protein expression by useof immunohistochemical means. Normal epithelial cells have a typicalapical distribution of MUC1, in addition to some supranuclearlocalization of the glycoprotein, whereas malignant lesions oftendemonstrate an apolar staining pattern (Diaz et al, The Breast Journal,7; 40-45 (2001); Zhang et al, Clinical Cancer Research, 4; 2669-2676(1998): Cao, et al, The Journal of Histochemistry and Cytochemistry, 45:1547-1557 (1997)). In addition, normal breast epithelium is eithernegative for Her2 protein or exhibits only a basolateral distributionwhereas malignant cells can express the protein over the whole cellsurface (De Potter, et al, International Journal of Cancer, 44; 969-974(1989): McCormick, et al, 117; 935-943 (2002)). Alternatively,distribution of the protein may be altered from a surface onlylocalization to include diffuse cytoplasmic expression in the diseasedstate. Such an example can be seen with MUC1 (Diaz, et al, The BreastJournal, 7: 40-45 (2001)).

Alteration in the localization/distribution of a protein in the cell, asdetected by immunohistochemical methods, can also provide valuableinformation concerning the favorability of certain treatment modalities.This last point is illustrated by a situation where a protein may beintracellular in normal tissue, but cell surface in malignant cells; thecell surface location makes the cells favorably amenable toantibody-based diagnostic and treatment regimens. When such analteration of protein localization occurs for 158P3D2, the 158P3D2protein and immune responses related thereto are very useful.Accordingly, the ability to determine whether alteration of subcellularprotein localization occurred for 24P4C 12 make the 158P3D2 protein andimmune responses related thereto very useful. Use of the 158P3D2compositions allows those skilled in the art to make importantdiagnostic and therapeutic decisions.

Immunohistochemical reagents specific to 158P3D2 are also useful todetect metastases of tumors expressing 158P3D2 when the polypeptideappears in tissues where 158P3D2 is not normally produced.

Thus, 158P3D2 polypeptides and antibodies resulting from immuneresponses thereto are useful in a variety of important contexts such asdiagnostic, prognostic, preventative and/or therapeutic purposes knownto those skilled in the art.

Just as PSA polynucleotide fragments and polynucleotide variants areemployed by skilled artisans for use in methods of monitoring PSA,158P3D2 polynucleotide fragments and polynucleotide variants are used inan analogous manner. In particular, typical PSA polynucleotides used inmethods of monitoring PSA are probes or primers which consist offragments of the PSA cDNA sequence. Illustrating this, primers used toPCR amplify a PSA polynucleotide must include less than the whole PSAsequence to function in the polymerase chain reaction. In the context ofsuch PCR reactions, skilled artisans generally create a variety ofdifferent polynucleotide fragments that can be used as primers in orderto amplify different portions of a polynucleotide of interest or tooptimize amplification reactions (see, e.g., Caetano-Anolles, G.Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., MethodsMol. Biol. 98:121-154 (1998)). An additional illustration of the use ofsuch fragments is provided in the Example entitled “Expression analysisof 158P3D2 in normal tissues, and patient specimens,” where a 158P3D2polynucleotide fragment is used as a probe to show the expression of158P3D2 RNAs in cancer cells. In addition, variant polynucleotidesequences are typically used as primers and probes for the correspondingmRNAs in PCR and Northern analyses (see, e.g., Sawai et al., FetalDiagn. Ther. 1996 Nov.-Dec. 11 (6):407-13 and Current Protocols InMolecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds.,1995)). Polynucleotide fragments and variants are useful in this contextwhere they are capable of binding to a target polynucleotide sequence(e.g., a 158P3D2 polynucleotide shown in FIG. 2 or variant thereof)under conditions of high stringency.

Furthermore, PSA polypeptides which contain an epitope that can berecognized by an antibody or T cell that specifically binds to thatepitope are used in methods of monitoring PSA. 158P3D2 polypeptidefragments and polypeptide analogs or variants can also be used in ananalogous manner. This practice of using polypeptide fragments orpolypeptide variants to generate antibodies (such as anti-PSA antibodiesor T cells) is typical in the art with a wide variety of systems such asfusion proteins being used by practitioners (see, e.g., CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubelet al. eds., 1995). In this context, each epitope(s) functions toprovide the architecture with which an antibody or T cell is reactive.Typically, skilled artisans create a variety of different polypeptidefragments that can be used in order to generate immune responsesspecific for different portions of a polypeptide of interest (see, e.g.,U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it maybe preferable to utilize a polypeptide comprising one of the 158P3D2biological motifs discussed herein or a motif-bearing subsequence whichis readily identified by one of skill in the art based on motifsavailable in the art. Polypeptide fragments, variants or analogs aretypically useful in this context as long as they comprise an epitopecapable of generating an antibody or T cell specific for a targetpolypeptide sequence (e.g. a 158P3D2 polypeptide shown in FIG. 3).

As shown herein, the 158P3D2 polynucleotides and polypeptides (as wellas the 158P3D2 polynucleotide probes and anti-158P3D2 antibodies or Tcells used to identify the presence of these molecules) exhibit specificproperties that make them useful in diagnosing cancers such as thoselisted in Table I. Diagnostic assays that measure the presence of158P3D2 gene products, in order to evaluate the presence or onset of adisease condition described herein, such as prostate cancer, are used toidentify patients for preventive measures or further monitoring, as hasbeen done so successfully with PSA. Moreover, these materials satisfy aneed in the art for molecules having similar or complementarycharacteristics to PSA in situations where, for example, a definitediagnosis of metastasis of prostatic origin cannot be made on the basisof a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract.192(3): 233-237 (1996)), and consequently, materials such as 158P3D2polynucleotides and polypeptides (as well as the 158P3D2 polynucleotideprobes and anti-158P3D2 antibodies used to identify the presence ofthese molecules) need to be employed to confirm a metastases ofprostatic origin.

Finally, in addition to their use in diagnostic assays, the 158P3D2polynucleotides disclosed herein have a number of other utilities suchas their use in the identification of oncogenetic associated chromosomalabnormalities in the chromosomal region to which the 158P3D2 gene maps(see the Example entitled “Chromosomal Mapping of 158P3D2” below).Moreover, in addition to their use in diagnostic assays, the158P3D2-related proteins and polynucleotides disclosed herein have otherutilities such as their use in the forensic analysis of tissues ofunknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun. 28;80(1-2): 63-9).

Additionally, 158P3D2-related proteins or polynucleotides of theinvention can be used to treat a pathologic condition characterized bythe over-expression of 158P3D2. For example, the amino acid or nucleicacid sequence of FIG. 2 or FIG. 3, or fragments of either, can be usedto generate an immune response to a 158P3D2 antigen. Antibodies or othermolecules that react with 158P3D2 can be used to modulate the functionof this molecule, and thereby provide a therapeutic benefit.

XII.) Inhibition of 158P3D2 Protein Function

The invention includes various methods and compositions for inhibitingthe binding of 158P3D2 to its binding partner or its association withother protein(s) as well as methods for inhibiting 158P3D2 function.

XII.A.) Inhibition of 158P3D2 with Intracellular Antibodies

In one approach, a recombinant vector that encodes single chainantibodies that specifically bind to 158P3D2 are introduced into 158P3D2expressing cells via gene transfer technologies. Accordingly, theencoded single chain anti-158P3D2 antibody is expressed intracellularly,binds to 158P3D2 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, arespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatment isfocused. This technology has been successfully applied in the art (forreview, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodieshave been shown to virtually eliminate the expression of otherwiseabundant cell surface receptors (see, e.g., Richardson et al., 1995,Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol.Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies areexpressed as a single chain variable region fragment joined to the lightchain constant region. Well-known intracellular trafficking signals areengineered into recombinant polynucleotide vectors encoding such singlechain antibodies in order to target precisely the intrabody to thedesired intracellular compartment. For example, intrabodies targeted tothe endoplasmic reticulum (ER) are engineered to incorporate a leaderpeptide and, optionally, a C-terminal ER retention signal, such as theKDEL amino acid motif. Intrabodies intended to exert activity in thenucleus are engineered to include a nuclear localization signal. Lipidmoieties are joined to intrabodies in order to tether the intrabody tothe cytosolic side of the plasma membrane. Intrabodies can also betargeted to exert function in the cytosol. For example, cytosolicintrabodies are used to sequester factors within the cytosol, therebypreventing them from being transported to their natural cellulardestination.

In one embodiment, intrabodies are used to capture 158P3D2 in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals are engineered into such 158P3D2 intrabodies in orderto achieve the desired targeting. Such 158P3D2 intrabodies are designedto bind specifically to a particular 158P3D2 domain. In anotherembodiment, cytosolic intrabodies that specifically bind to a 158P3D2protein are used to prevent 158P3D2 from gaining access to the nucleus,thereby preventing it from exerting any biological activity within thenucleus (e.g., preventing 158P3D2 from forming transcription complexeswith other factors).

In order to specifically direct the expression of such intrabodies toparticular cells, the transcription of the intrabody is placed under theregulatory control of an appropriate tumor-specific promoter and/orenhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer can beutilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).

XII.B.) Inhibition of 158P3D2 with Recombinant Proteins

In another approach, recombinant molecules bind to 158P3D2 and therebyinhibit 158P3D2 function. For example, these recombinant moleculesprevent or inhibit 158P3D2 from accessing/binding to its bindingpartner(s) or associating with other protein(s). Such recombinantmolecules can, for example, contain the reactive part(s) of a 158P3D2specific antibody molecule. In a particular embodiment, the 158P3D2binding domain of a 158P3D2 binding partner is engineered into a dimericfusion protein, whereby the fusion protein comprises two 158P3D2 ligandbinding domains linked to the Fc portion of a human IgG, such as humanIgG1. Such IgG portion can contain, for example, the CH2 and CH3 domainsand the hinge region, but not the CH1 domain. Such dimeric fusionproteins are administered in soluble form to patients suffering from acancer associated with the expression of 158P3D2, whereby the dimericfusion protein specifically binds to 158P3D2 and blocks 158P3D2interaction with a binding partner. Such dimeric fusion proteins arefurther combined into multimeric proteins using known antibody linkingtechnologies.

XII.C.) Inhibition of 158P3D2 Transcription or Translation

The present invention also comprises various methods and compositionsfor inhibiting the transcription of the 158P3D2 gene. Similarly, theinvention also provides methods and compositions for inhibiting thetranslation of 158P3D2 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 158P3D2gene comprises contacting the 158P3D2 gene with a 158P3D2 antisensepolynucleotide. In another approach, a method of inhibiting 158P3D2 mRNAtranslation comprises contacting a 158P3D2 mRNA with an antisensepolynucleotide. In another approach, a 158P3D2 specific ribozyme is usedto cleave a 158P3D2 message, thereby inhibiting translation. Suchantisense and ribozyme based methods can also be directed to theregulatory regions of the 158P3D2 gene, such as 158P3D2 promoter and/orenhancer elements. Similarly, proteins capable of inhibiting a 158P3D2gene transcription factor are used to inhibit 158P3D2 mRNAtranscription. The various polynucleotides and compositions useful inthe aforementioned methods have been described above. The use ofantisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

Other factors that inhibit the transcription of 158P3D2 by interferingwith 158P3D2 transcriptional activation are also useful to treat cancersexpressing 158P3D2. Similarly, factors that interfere with 158P3D2processing are useful to treat cancers that express 158P3D2. Cancertreatment methods utilizing such factors are also within the scope ofthe invention.

XII.D.) General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to delivertherapeutic polynucleotide molecules to tumor cells synthesizing 158P3D2(i.e., antisense, ribozyme, polynucleotides encoding intrabodies andother 158P3D2 inhibitory molecules). A number of gene therapy approachesare known in the art. Recombinant vectors encoding 158P3D2 antisensepolynucleotides, ribozymes, factors capable of interfering with 158P3D2transcription, and so forth, can be delivered to target tumor cellsusing such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a widevariety of surgical, chemotherapy or radiation therapy regimens. Thetherapeutic approaches of the invention can enable the use of reduceddosages of chemotherapy (or other therapies) and/or less frequentadministration, an advantage for all patients and particularly for thosethat do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, can beevaluated using various in vitro and in vivo assay systems. In vitroassays that evaluate therapeutic activity include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 158P3D2 to a bindingpartner, etc.

In vivo, the effect of a 158P3D2 therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic prostatecancer models can be used, wherein human prostate cancer explants orpassaged xenograft tissues are introduced into immune compromisedanimals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine3: 402-408). For example, PCT Patent Application WO98/16628 and U.S.Pat. No. 6,107,540 describe various xenograft models of human prostatecancer capable of recapitulating the development of primary tumors,micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy can be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful inevaluating therapeutic compositions. In one embodiment, xenografts fromtumor bearing mice treated with the therapeutic composition can beexamined for the presence of apoptotic foci and compared to untreatedcontrol xenograft-bearing mice. The extent to which apoptotic foci arefound in the tumors of the treated mice provides an indication of thetherapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Therapeutic formulations can be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancer,and will generally depend on a number of other factors appreciated inthe art.

XIII.) Identification, Characterization and Use of Modulators of 158P3D2

XIII.A.) Methods to Identify and Use Modulators

In one embodiment, screening is performed to identify modulators thatinduce or suppress a particular expression profile, suppress or inducespecific pathways, preferably generating the associated phenotypethereby. In another embodiment, having identified differentiallyexpressed genes important in a particular state; screens are performedto identify modulators that alter expression of individual genes, eitherincrease or decrease. In another embodiment, screening is performed toidentify modulators that alter a biological function of the expressionproduct of a differentially expressed gene. Again, having identified theimportance of a gene in a particular state, screens are performed toidentify agents that bind and/or modulate the biological activity of thegene product.

In addition, screens are done for genes that are induced in response toa candidate agent. After identifying a modulator (one that suppresses acancer expression pattern leading to a normal expression pattern, or amodulator of a cancer gene that leads to expression of the gene as innormal tissue) a screen is performed to identify genes that arespecifically modulated in response to the agent. Comparing expressionprofiles between normal tissue and agent-treated cancer tissue revealsgenes that are not expressed in normal tissue or cancer tissue, but areexpressed in agent treated tissue, and vice versa. These agent-specificsequences are identified and used by methods described herein for cancergenes or proteins. In particular these sequences and the proteins theyencode are used in marking or identifying agent-treated cells. Inaddition, antibodies are raised against the agent-induced proteins andused to target novel therapeutics to the treated cancer tissue sample.

XIII.B.) Gene Expression-Related Assays

Proteins, nucleic acids, and antibodies of the invention are used inscreening assays. The cancer-associated proteins, antibodies, nucleicacids, modified proteins and cells containing these sequences are usedin screening assays, such as evaluating the effect of drug candidates ona “gene expression profile,” expression profile of polypeptides oralteration of biological function. In one embodiment, the expressionprofiles are used, preferably in conjunction with high throughputscreening techniques to allow monitoring for expression profile genesafter treatment with a candidate agent (e.g., Davis, G F, et al, J BiolScreen 7:69 (2002); Zlokarnik, et al., Science 279:84-8 (1998); Heid,Genome Res 6:986-94,1996).

The cancer proteins, antibodies, nucleic acids, modified proteins andcells containing the native or modified cancer proteins or genes areused in screening assays. That is, the present invention comprisesmethods for screening for compositions which modulate the cancerphenotype or a physiological function of a cancer protein of theinvention. This is done on a gene itself or by evaluating the effect ofdrug candidates on a “gene expression profile” or biological function.In one embodiment, expression profiles are used, preferably inconjunction with high throughput screening techniques to allowmonitoring after treatment with a candidate agent, see Zlokamik, supra.

A variety of assays are executed directed to the genes and proteins ofthe invention. Assays are run on an individual nucleic acid or proteinlevel. That is, having identified a particular gene as up regulated incancer, test compounds are screened for the ability to modulate geneexpression or for binding to the cancer protein of the invention.“Modulation” in this context includes an increase or a decrease in geneexpression. The preferred amount of modulation will depend on theoriginal change of the gene expression in normal versus tissueundergoing cancer, with changes of at least 10%, preferably 50%, morepreferably 100-300%, and in some embodiments 300-1000% or greater. Thus,if a gene exhibits a 4-fold increase in cancer tissue compared to normaltissue, a decrease of about four-fold is often desired; similarly, a10-fold decrease in cancer tissue compared to normal tissue a targetvalue of a 10-fold increase in expression by the test compound is oftendesired. Modulators that exacerbate the type of gene expression seen incancer are also useful, e.g., as an upregulated target in furtheranalyses.

The amount of gene expression is monitored using nucleic acid probes andthe quantification of gene expression levels, or, alternatively, a geneproduct itself is monitored, e.g., through the use of antibodies to thecancer protein and standard immunoassays. Proteomics and separationtechniques also allow for quantification of expression.

XIII.C.) Expression Monitoring to Identify Compounds that Modify GeneExpression

In one embodiment, gene expression monitoring, i.e., an expressionprofile, is monitored simultaneously for a number of entities. Suchprofiles will typically involve one or more of the genes of FIG. 2. Inthis embodiment, e.g., cancer nucleic acid probes are attached tobiochips to detect and quantify cancer sequences in a particular cell.Alternatively, PCR can be used. Thus, a series, e.g., wells of amicrotiter plate, can be used with dispensed primers in desired wells. APCR reaction can then be performed and analyzed for each well.

Expression monitoring is performed to identify compounds that modify theexpression of one or more cancer-associated sequences, e.g., apolynucleotide sequence set out in FIG. 2. Generally, a test modulatoris added to the cells prior to analysis. Moreover, screens are alsoprovided to identify agents that modulate cancer, modulate cancerproteins of the invention, bind to a cancer protein of the invention, orinterfere with the binding of a cancer protein of the invention and anantibody or other binding partner.

In one embodiment, high throughput screening methods involve providing alibrary containing a large number of potential therapeutic compounds(candidate compounds). Such “combinatorial chemical libraries” are thenscreened in one or more assays to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds thus identified can serve asconventional “lead compounds,” as compounds for screening, or astherapeutics.

In certain embodiments, combinatorial libraries of potential modulatorsare screened for an ability to bind to a cancer polypeptide or tomodulate activity. Conventionally, new chemical entities with usefulproperties are generated by identifying a chemical compound (called a“lead compound”) with some desirable property or activity, e.g.,inhibiting activity, creating variants of the lead compound, andevaluating the property and activity of those variant compounds. Often,high throughput screening (HTS) methods are employed for such ananalysis.

As noted above, gene expression monitoring is conveniently used to testcandidate modulators (e.g., protein, nucleic acid or small molecule).After the candidate agent has been added and the cells allowed toincubate for a period, the sample containing a target sequence to beanalyzed is, e.g., added to a biochip.

If required, the target sequence is prepared using known techniques. Forexample, a sample is treated to lyse the cells, using known lysisbuffers, electroporation, etc., with purification and/or amplificationsuch as PCR performed as appropriate. For example, an in vitrotranscription with labels covalently attached to the nucleotides isperformed. Generally, the nucleic acids are labeled with biotin-FITC orPE, or with cy3 or cy5.

The target sequence can be labeled with, e.g., a fluorescent, achemiluminescent, a chemical, or a radioactive signal, to provide ameans of detecting the target sequence's specific binding to a probe.The label also can be an enzyme, such as alkaline phosphatase orhorseradish peroxidase, which when provided with an appropriatesubstrate produces a product that is detected. Alternatively, the labelis a labeled compound or small molecule, such as an enzyme inhibitor,that binds but is not catalyzed or altered by the enzyme. The label alsocan be a moiety or compound, such as, an epitope tag or biotin whichspecifically binds to streptavidin. For the example of biotin, thestreptavidin is labeled as described above, thereby, providing adetectable signal for the bound target sequence. Unbound labeledstreptavidin is typically removed prior to analysis.

As will be appreciated by those in the art, these assays can be directhybridization assays or can comprise “sandwich assays”, which includethe use of multiple probes, as is generally outlined in U.S. Pat. Nos.5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670;5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118;5,359,100; 5,124,246; and 5,681,697. In this embodiment, in general, thetarget nucleic acid is prepared as outlined above, and then added to thebiochip comprising a plurality of nucleic acid probes, under conditionsthat allow the formation of a hybridization complex.

A variety of hybridization conditions are used in the present invention,including high, moderate and low stringency conditions as outlinedabove. The assays are generally run under stringency conditions whichallow formation of the label probe hybridization complex only in thepresence of target. Stringency can be controlled by altering a stepparameter that is a thermodynamic variable, including, but not limitedto, temperature, formamide concentration, salt concentration, chaotropicsalt concentration pH, organic solvent concentration, etc. Theseparameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus, it can be desirableto perform certain steps at higher stringency conditions to reducenon-specific binding.

The reactions outlined herein can be accomplished in a variety of ways.Components of the reaction can be added simultaneously, or sequentially,in different orders, with preferred embodiments outlined below. Inaddition, the reaction may include a variety of other reagents. Theseinclude salts, buffers, neutral proteins, e.g. albumin, detergents, etc.which can be used to facilitate optimal hybridization and detection,and/or reduce nonspecific or background interactions. Reagents thatotherwise improve the efficiency of the assay, such as proteaseinhibitors, nuclease inhibitors, anti-microbial agents, etc., may alsobe used as appropriate, depending on the sample preparation methods andpurity of the target. The assay data are analyzed to determine theexpression levels of individual genes, and changes in expression levelsas between states, forming a gene expression profile.

XIII.D.) Biological Activity-Related Assays

The invention provides methods identify or screen for a compound thatmodulates the activity of a cancer-related gene or protein of theinvention. The methods comprise adding a test compound, as definedabove, to a cell comprising a cancer protein of the invention. The cellscontain a recombinant nucleic acid that encodes a cancer protein of theinvention. In another embodiment, a library of candidate agents istested on a plurality of cells.

In one aspect, the assays are evaluated in the presence or absence orprevious or subsequent exposure of physiological signals, e.g. hormones,antibodies, peptides, antigens, cytokines, growth factors, actionpotentials, pharmacological agents including chemotherapeutics,radiation, carcinogenics, or other cells (i.e., cell-cell contacts). Inanother example, the determinations are made at different stages of thecell cycle process. In this way, compounds that modulate genes orproteins of the invention are identified. Compounds with pharmacologicalactivity are able to enhance or interfere with the activity of thecancer protein of the invention. Once identified, similar structures areevaluated to identify critical structural features of the compound.

In one embodiment, a method of modulating (e.g., inhibiting) cancer celldivision is provided; the method comprises administration of a cancermodulator. In another embodiment, a method of modulating (e.g.,inhibiting) cancer is provided; the method comprises administration of acancer modulator. In a further embodiment, methods of treating cells orindividuals with cancer are provided; the method comprisesadministration of a cancer modulator.

In one embodiment, a method for modulating the status of a cell thatexpresses a gene of the invention is provided. As used herein statuscomprises such art-accepted parameters such as growth, proliferation,survival, function, apoptosis, senescence, location, enzymatic activity,signal transduction, etc. of a cell. In one embodiment, a cancerinhibitor is an antibody as discussed above. In another embodiment, thecancer inhibitor is an antisense molecule. A variety of cell growth,proliferation, and metastasis assays are known to those of skill in theart, as described herein.

XIII.E.) High Throughput Screening to Identify Modulators

The assays to identify suitable modulators are amenable to highthroughput screening. Preferred assays thus detect enhancement orinhibition of cancer gene transcription, inhibition or enhancement ofpolypeptide expression, and inhibition or enhancement of polypeptideactivity.

In one embodiment, modulators evaluated in high throughput screeningmethods are proteins, often naturally occurring proteins or fragments ofnaturally occurring proteins. Thus, e.g., cellular extracts containingproteins, or random or directed digests of proteinaceous cellularextracts, are used. In this way, libraries of proteins are made forscreening in the methods of the invention. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred. Particularly useful test compound will be directedto the class of proteins to which the target belongs, e.g., substratesfor enzymes, or ligands and receptors.

XIII.F.) Use of Soft Agar Growth and Colony Formation to Identify andCharacterize Modulators

Normal cells require a solid substrate to attach and grow. When cellsare transformed, they lose this phenotype and grow detached from thesubstrate. For example, transformed cells can grow in stirred suspensionculture or suspended in semi-solid media, such as semi-solid or softagar. The transformed cells, when transfected with tumor suppressorgenes, can regenerate normal phenotype and once again require a solidsubstrate to attach to and grow. Soft agar growth or colony formation inassays are used to identify modulators of cancer sequences, which whenexpressed in host cells, inhibit abnormal cellular proliferation andtransformation. A modulator reduces or eliminates the host cells'ability to grow suspended in solid or semisolid media, such as agar.

Techniques for soft agar growth or colony formation in suspension assaysare described in Freshney, Culture of Animal Cells a Manual of BasicTechnique (3rd ed., 1994). See also, the methods section of Garkavtsevet al. (1996), supra.

XIII.G.) Evaluation of Contact Inhibition and Growth Density Limitationto Identify and Characterize Modulators

Normal cells typically grow in a flat and organized pattern in cellculture until they touch other cells. When the cells touch one another,they are contact inhibited and stop growing. Transformed cells, however,are not contact inhibited and continue to grow to high densities indisorganized foci. Thus, transformed cells grow to a higher saturationdensity than corresponding normal cells. This is detectedmorphologically by the formation of a disoriented monolayer of cells orcells in foci. Alternatively, labeling index with (3H)-thymidine atsaturation density is used to measure density limitation of growth,similarly an MTT or Alamar blue assay will reveal proliferation capacityof cells and the the ability of modulators to affect same. See Freshney(1994), supra. Transformed cells, when transfected with tumor suppressorgenes, can regenerate a normal phenotype and become contact inhibitedand would grow to a lower density.

In this assay, labeling index with 3H)-thymidine at saturation densityis a preferred method of measuring density limitation of growth.Transformed host cells are transfected with a cancer-associated sequenceand are grown for 24 hours at saturation density in non-limiting mediumconditions. The percentage of cells labeling with (3H)-thymidine isdetermined by incorporated cpm.

Contact independent growth is used to identify modulators of cancersequences, which had led to abnormal cellular proliferation andtransformation. A modulator reduces or eliminates contact independentgrowth, and returns the cells to a normal phenotype.

XIII.H.) Evaluation of Growth Factor or Serum Dependence to Identify andCharacterize Modulators

Transformed cells have lower serum dependence than their normalcounterparts (see, e.g., Temin, J. Natl. Cancer Inst. 37:167-175 (1966);Eagle et al., J. Exp. Med 131:836-879 (1970)); Freshney, supra. This isin part due to release of various growth factors by the transformedcells. The degree of growth factor or serum dependence of transformedhost cells can be compared with that of control. For example, growthfactor or serum dependence of a cell is monitored in methods to identifyand characterize compounds that modulate cancer-associated sequences ofthe invention.

XIII.I.) Use of Tumor-Specific Marker Levels to Identify andCharacterize Modulators

Tumor cells release an increased amount of certain factors (hereinafter“tumor specific markers”) than their normal counterparts. For example,plasminogen activator (PA) is released from human glioma at a higherlevel than from normal brain cells (see, e.g., Gullino, Angiogenesis,Tumor Vascularization, and Potential Interference with Tumor Growth, inBiological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)).Similarly, Tumor Angiogenesis Factor (TAF) is released at a higher levelin tumor cells than their normal counterparts. See, e.g., Folkman,Angiogenesis and Cancer, Sem Cancer Biol. (1992)), while bFGF isreleased from endothelial tumors (Ensoli, B et al).

Various techniques which measure the release of these factors aredescribed in Freshney (1994), supra. Also, see, Unkless et al., J. Biol.Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem.251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305 312 (1980);Gullino, Angiogenesis, Tumor Vascularization, and Potential Interferencewith Tumor Growth, in Biological Responses in Cancer, pp. 178-184(Mihich (ed.) 1985); Freshney, Anticancer Res. 5:111-130 (1985). Forexample, tumor specific marker levels are monitored in methods toidentify and characterize compounds that modulate cancer-associatedsequences of the invention.

XIII.J.) Invasiveness into Matrigel to Identify and CharacterizeModulators

The degree of invasiveness into Matrigel or an extracellular matrixconstituent can be used as an assay to identify and characterizecompounds that modulate cancer associated sequences. Tumor cells exhibita positive correlation between malignancy and invasiveness of cells intoMatrigel or some other extracellular matrix constituent. In this assay,tumorigenic cells are typically used as host cells. Expression of atumor suppressor gene in these host cells would decrease invasiveness ofthe host cells. Techniques described in Cancer Res. 1999; 59:6010;Freshney (1994), supra, can be used. Briefly, the level of invasion ofhost cells is measured by using filters coated with Matrigel or someother extracellular matrix constituent. Penetration into the gel, orthrough to the distal side of the filter, is rated as invasiveness, andrated histologically by number of cells and distance moved, or byprelabeling the cells with 1251 and counting the radioactivity on thedistal side of the filter or bottom of the dish. See, e.g., Freshney(1984), supra.

XIII.K.) Evaluation of Tumor Growth In Vivo to Identify and CharacterizeModulators

Effects of cancer-associated sequences on cell growth are tested intransgenic or immune-suppressed organisms. Transgenic organisms areprepared in a variety of art-accepted ways. For example, knock-outtransgenic organisms, e.g., mammals such as mice, are made, in which acancer gene is disrupted or in which a cancer gene is inserted.Knock-out transgenic mice are made by insertion of a marker gene orother heterologous gene into the endogenous cancer gene site in themouse genome via homologous recombination. Such mice can also be made bysubstituting the endogenous cancer gene with a mutated version of thecancer gene, or by mutating the endogenous cancer gene, e.g., byexposure to carcinogens.

To prepare transgenic chimeric animals, e.g., mice, a DNA construct isintroduced into the nuclei of embryonic stem cells. Cells containing thenewly engineered genetic lesion are injected into a host mouse embryo,which is re-implanted into a recipient female. Some of these embryosdevelop into chimeric mice that possess germ cells some of which arederived from the mutant cell line. Therefore, by breeding the chimericmice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288(1989)). Chimeric mice can be derived according to U.S. Pat. No.6,365,797, issued 2 Apr. 2002; U.S. Pat. No. 6,107,540 issued 22 Aug.2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual,Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and EmbryonicStem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington,D.C., (1987).

Alternatively, various immune-suppressed or immune-deficient hostanimals can be used. For example, a genetically athymic “nude” mouse(see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), aSCID mouse, a thymectornized mouse, or an irradiated mouse (see, e.g.,Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer41:52 (1980)) can be used as a host. Transplantable tumor cells(typically about 106 cells) injected into isogenic hosts produceinvasive tumors in a high proportion of cases, while normal cells ofsimilar origin will not. In hosts which developed invasive tumors, cellsexpressing cancer-associated sequences are injected subcutaneously ororthotopically. Mice are then separated into groups, including controlgroups and treated experimental groups) e.g. treated with a modulator).After a suitable length of time, preferably 4-8 weeks, tumor growth ismeasured (e.g., by volume or by its two largest dimensions, or weight)and compared to the control. Tumors that have statistically significantreduction (using, e.g., Student's T test) are said to have inhibitedgrowth.

XIII.L.) In Vitro Assays to Identify and Characterize Modulators

Assays to identify compounds with modulating activity can be performedin vitro. For example, a cancer polypeptide is first contacted with apotential modulator and incubated for a suitable amount of time, e.g.,from 0.5 to 48 hours. In one embodiment, the cancer polypeptide levelsare determined in vitro by measuring the level of protein or mRNA. Thelevel of protein is measured using immunoassays such as Westernblotting, ELISA and the like with an antibody that selectively binds tothe cancer polypeptide or a fragment thereof. For measurement of mRNA,amplification, e.g., using PCR, LCR, or hybridization assays, e.g.,Northern hybridization, RNAse protection, dot blotting, are preferred.The level of protein or mRNA is detected using directly or indirectlylabeled detection agents, e.g., fluorescently or radioactively labelednucleic acids, radioactively or enzymatically labeled antibodies, andthe like, as described herein.

Alternatively, a reporter gene system can be devised using a cancerprotein promoter operably linked to a reporter gene such as luciferase,green fluorescent protein, CAT, or P-gal. The reporter construct istypically transfected into a cell. After treatment with a potentialmodulator, the amount of reporter gene transcription, translation, oractivity is measured according to standard techniques known to those ofskill in the art (Davis G F, supra; Gonzalez, J. & Negulescu, P. Curr.Opin. Biotechnol. 1998: 9:624).

As outlined above, in vitro screens are done on individual genes andgene products. That is, having identified a particular differentiallyexpressed gene as important in a particular state, screening ofmodulators of the expression of the gene or the gene product itself isperformed.

In one embodiment, screening for modulators of expression of specificgene(s) is performed. Typically, the expression of only one or a fewgenes is evaluated. In another embodiment, screens are designed to firstfind compounds that bind to differentially expressed proteins. Thesecompounds are then evaluated for the ability to modulate differentiallyexpressed activity. Moreover, once initial candidate compounds areidentified, variants can be further screened to better evaluatestructure activity relationships.

XIII.M.) Binding Assays to Identify and Characterize Modulators

In binding assays in accordance with the invention, a purified orisolated gene product of the invention is generally used. For example,antibodies are generated to a protein of the invention, and immunoassaysare run to determine the amount and/or location of protein.Alternatively, cells comprising the cancer proteins are used in theassays.

Thus, the methods comprise combining a cancer protein of the inventionand a candidate compound such as a ligand, and determining the bindingof the compound to the cancer protein of the invention. Preferredembodiments utilize the human cancer protein; animal models of humandisease of can also be developed and used. Also, other analogousmammalian proteins also can be used as appreciated by those of skill inthe art. Moreover, in some embodiments variant or derivative cancerproteins are used.

Generally, the cancer protein of the invention, or the ligand, isnon-diffusibly bound to an insoluble support. The support can, e.g., beone having isolated sample receiving areas (a microtiter plate, anarray, etc.). The insoluble supports can be made of any composition towhich the compositions can be bound, is readily separated from solublematerial, and is otherwise compatible with the overall method ofscreening. The surface of such supports can be solid or porous and ofany convenient shape.

Examples of suitable insoluble supports include microtiter plates,arrays, membranes and beads. These are typically made of glass, plastic(e.g., polystyrene), polysaccharide, nylon, nitrocellulose, or Teflon™,etc. Microtiter plates and arrays are especially convenient because alarge number of assays can be carried out simultaneously, using smallamounts of reagents and samples. The particular manner of binding of thecomposition to the support is not crucial so long as it is compatiblewith the reagents and overall methods of the invention, maintains theactivity of the composition and is nondiffusable. Preferred methods ofbinding include the use of antibodies which do not sterically blockeither the ligand binding site or activation sequence when attaching theprotein to the support, direct binding to “sticky” or ionic supports,chemical crosslinking, the synthesis of the protein or agent on thesurface, etc. Following binding of the protein or ligand/binding agentto the support, excess unbound material is removed by washing. Thesample receiving areas may then be blocked through incubation withbovine serum albumin (BSA), casein or other innocuous protein or othermoiety.

Once a cancer protein of the invention is bound to the support, and atest compound is added to the assay. Alternatively, the candidatebinding agent is bound to the support and the cancer protein of theinvention is then added. Binding agents include specific antibodies,non-natural binding agents identified in screens of chemical libraries,peptide analogs, etc.

Of particular interest are assays to identify agents that have a lowtoxicity for human cells. A wide variety of assays can be used for thispurpose, including proliferation assays, cAMP assays, labeled in vitroprotein-protein binding assays, electrophoretic mobility shift assays,immunoassays for protein binding, functional assays (phosphorylationassays, etc.) and the like.

A determination of binding of the test compound (ligand, binding agent,modulator, etc.) to a cancer protein of the invention can be done in anumber of ways. The test compound can be labeled, and binding determineddirectly, e.g., by attaching all or a portion of the cancer protein ofthe invention to a solid support, adding a labeled candidate compound(e.g., a fluorescent label), washing off excess reagent, and determiningwhether the label is present on the solid support. Various blocking andwashing steps can be utilized as appropriate.

In certain embodiments, only one of the components is labeled, e.g., aprotein of the invention or ligands labeled. Alternatively, more thanone component is labeled with different labels, e.g., 1125, for theproteins and a fluorophor for the compound. Proximity reagents, e.g.,quenching or energy transfer reagents are also useful.

XIII.N.) Competitive Binding to Identify and Characterize Modulators

In one embodiment, the binding of the “test compound” is determined bycompetitive binding assay with a “competitor.” The competitor is abinding moiety that binds to the target molecule (e.g., a cancer proteinof the invention). Competitors include compounds such as antibodies,peptides, binding partners, ligands, etc. Under certain circumstances,the competitive binding between the test compound and the competitordisplaces the test compound. In one embodiment, the test compound islabeled. Either the test compound, the competitor, or both, is added tothe protein for a time sufficient to allow binding. Incubations areperformed at a temperature that facilitates optimal activity, typicallybetween four and 40° C. Incubation periods are typically optimized,e.g., to facilitate rapid high throughput screening; typically betweenzero and one hour will be sufficient. Excess reagent is generallyremoved or washed away. The second component is then added, and thepresence or absence of the labeled component is followed, to indicatebinding.

In one embodiment, the competitor is added first, followed by the testcompound. Displacement of the competitor is an indication that the testcompound is binding to the cancer protein and thus is capable of bindingto, and potentially modulating, the activity of the cancer protein. Inthis embodiment, either component can be labeled. Thus, e.g., if thecompetitor is labeled, the presence of label in the post-test compoundwash solution indicates displacement by the test compound.Alternatively, if the test compound is labeled, the presence of thelabel on the support indicates displacement.

In an alternative embodiment, the test compound is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor indicates that the test compound binds to thecancer protein with higher affinity than the competitor. Thus, if thetest compound is labeled, the presence of the label on the support,coupled with a lack of competitor binding, indicates that the testcompound binds to and thus potentially modulates the cancer protein ofthe invention.

Accordingly, the competitive binding methods comprise differentialscreening to identity agents that are capable of modulating the activityof the cancer proteins of the invention. In this embodiment, the methodscomprise combining a cancer protein and a competitor in a first sample.A second sample comprises a test compound, the cancer protein, and acompetitor. The binding of the competitor is determined for bothsamples, and a change, or difference in binding between the two samplesindicates the presence of an agent capable of binding to the cancerprotein and potentially modulating its activity. That is, if the bindingof the competitor is different in the second sample relative to thefirst sample, the agent is capable of binding to the cancer protein.

Alternatively, differential screening is used to identify drugcandidates that bind to the native cancer protein, but cannot bind tomodified cancer proteins. For example the structure of the cancerprotein is modeled and used in rational drug design to synthesize agentsthat interact with that site, agents which generally do not bind tosite-modified proteins. Moreover, such drug candidates that affect theactivity of a native cancer protein are also identified by screeningdrugs for the ability to either enhance or reduce the activity of suchproteins.

Positive controls and negative controls can be used in the assays.Preferably control and test samples are performed in at least triplicateto obtain statistically significant results. Incubation of all samplesoccurs for a time sufficient to allow for the binding of the agent tothe protein. Following incubation, samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples can be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents can be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc. which are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,can be used. The mixture of components is added in an order thatprovides for the requisite binding.

XIII.O.) Use of Polynucleotides to Down-Regulate or Inhibit a Protein ofthe Invention.

Polynucleotide modulators of cancer can be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand-binding molecule, as described in WO 91/04753. Suitableligand-binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell. Alternatively, a polynucleotide modulator ofcancer can be introduced into a cell containing the target nucleic acidsequence, e.g., by formation of a polynucleotide-lipid complex, asdescribed in WO 90/10448. It is understood that the use of antisensemolecules or knock out and knock in models may also be used in screeningassays as discussed above, in addition to methods of treatment.

XIII.P.) Inhibitory and Antisense Nucleotides

In certain embodiments, the activity of a cancer-associated protein isdown-regulated, or entirely inhibited, by the use of antisensepolynucleotide or inhibitory small nuclear RNA (snRNA), i.e., a nucleicacid complementary to, and which can preferably hybridize specificallyto, a coding mRNA nucleic acid sequence, e.g., a cancer protein of theinvention, mRNA, or a subsequence thereof. Binding of the antisensepolynucleotide to the mRNA reduces the translation and/or stability ofthe mRNA.

In the context of this invention, antisense polynucleotides can comprisenaturally occurring nucleotides, or synthetic species formed fromnaturally occurring subunits or their close homologs. Antisensepolynucleotides may also have altered sugar moieties or inter-sugarlinkages. Exemplary among these are the phosphorothioate and othersulfur containing species which are known for use in the art. Analogsare comprised by this invention so long as they function effectively tohybridize with nucleotides of the invention. See, e.g., IsisPharmaceuticals, Carlsbad, Calif.; Sequitor, Inc., Natick, Mass.

Such antisense polynucleotides can readily be synthesized usingrecombinant means, or can be synthesized in vitro. Equipment for suchsynthesis is sold by several vendors, including Applied Biosystems. Thepreparation of other oligonucleotides such as phosphorothioates andalkylated derivatives is also well known to those of skill in the art.

Antisense molecules as used herein include antisense or senseoligonucleotides. Sense oligonucleotides can, e.g., be employed to blocktranscription by binding to the anti-sense strand. The antisense andsense oligonucleotide comprise a single stranded nucleic acid sequence(either RNA or DNA) capable of binding to target mRNA (sense) or DNA(antisense) sequences for cancer molecules. Antisense or senseoligonucleotides, according to the present invention, comprise afragment generally at least about 12 nucleotides, preferably from about12 to 30 nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, e.g., Stein &Cohen (Cancer Res. 48:2659 (1988 and van derKrol et al. (BioTechniques 6:958 (1988)).

XIII.Q.) Ribozymes

In addition to antisense polynucleotides, ribozymes can be used totarget and inhibit transcription of cancer-associated nucleotidesequences. A ribozyme is an RNA molecule that catalytically cleavesother RNA molecules. Different kinds of ribozymes have been described,including group I ribozymes, hammerhead ribozymes, hairpin ribozymes,RNase P, and axhead ribozymes (see, e.g., Castanotto et al., Adv. inPharmacology 25: 289-317 (1994) for a general review of the propertiesof different ribozymes).

The general features of hairpin ribozymes are described, e.g., in Hampelet al., Nucl. Acids Res. 18:299-304 (1990); European Patent PublicationNo. 0360257; U.S. Pat. No. 5,254,678. Methods of preparing are wellknown to those of skill in the art (see, e.g., WO 94/26877; Ojwang etal., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al.,Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad.Sci. USA 92:699-703 (1995); Leavitt et al., Human Gene Therapy 5:1151-120 (1994); and Yamada et al., Virology 205: 121-126 (1994)).

XIII.R.) Use of Modulators in Phenotypic Screening

In one embodiment, a test compound is administered to a population ofcancer cells, which have an associated cancer expression profile. By“administration” or “contacting” herein is meant that the modulator isadded to the cells in such a manner as to allow the modulator to actupon the cell, whether by uptake and intracellular action, or by actionat the cell surface. In some embodiments, a nucleic acid encoding aproteinaceous agent (i.e., a peptide) is put into a viral construct suchas an adenoviral or retroviral construct, and added to the cell, suchthat expression of the peptide agent is accomplished, e.g., PCTUS97/01019. Regulatable gene therapy systems can also be used. Once themodulator has been administered to the cells, the cells are washed ifdesired and are allowed to incubate under preferably physiologicalconditions for some period. The cells are then harvested and a new geneexpression profile is generated. Thus, e.g., cancer tissue is screenedfor agents that modulate, e.g., induce or suppress, the cancerphenotype. A change in at least one gene, preferably many, of theexpression profile indicates that the agent has an effect on canceractivity. Similarly, altering a biological function or a signalingpathway is indicative of modulator activity. By defining such asignature for the cancer phenotype, screens for new drugs that alter thephenotype are devised. With this approach, the drug target need not beknown and need not be represented in the original gene/proteinexpression screening platform, nor does the level of transcript for thetarget protein need to change. The modulator inhibiting function willserve as a surrogate marker.

As outlined above, screens are done to assess genes or gene products.That is, having identified a particular differentially expressed gene asimportant in a particular state, screening of modulators of either theexpression of the gene or the gene product itself is performed.

XIII.S.) Use of Modulators to Affect Peptides of the Invention

Measurements of cancer polypeptide activity, or of the cancer phenotypeare performed using a variety of assays. For example, the effects ofmodulators upon the function of a cancer polypeptide(s) are measured byexamining parameters described above. A physiological change thataffects activity is used to assess the influence of a test compound onthe polypeptides of this invention. When the functional outcomes aredetermined using intact cells or animals, a variety of effects can beassesses such as, in the case of a cancer associated with solid tumors,tumor growth, tumor metastasis, neovascularization, hormone release,transcriptional changes to both known and uncharacterized geneticmarkers (e.g., by Northern blots), changes in cell metabolism such ascell growth or pH changes, and changes in intracellular secondmessengers such as cGNIP.

XIII.T.) Methods of Identifying Characterizing Cancer-AssociatedSequences

Expression of various gene sequences is correlated with cancer.Accordingly, disorders based on mutant or variant cancer genes aredetermined. In one embodiment, the invention provides methods foridentifying cells containing variant cancer genes, e.g., determining thepresence of, all or part, the sequence of at least one endogenous cancergene in a cell. This is accomplished using any number of sequencingtechniques. The invention comprises methods of identifying the cancergenotype of an individual, e.g., determining all or part of the sequenceof at least one gene of the invention in the individual. This isgenerally done in at least one tissue of the individual, e.g., a tissueset forth in Table I, and may include the evaluation of a number oftissues or different samples of the same tissue. The method may includecomparing the sequence of the sequenced gene to a known cancer gene,i.e., a wild-type gene to determine the presence of family members,homologies, mutations or variants. The sequence of all or part of thegene can then be compared to the sequence of a known cancer gene todetermine if any differences exist. This is done using any number ofknown homology programs, such as BLAST, Bestfit, etc. The presence of adifference in the sequence between the cancer gene of the patient andthe known cancer gene correlates with a disease state or a propensityfor a disease state, as outlined herein.

In a preferred embodiment, the cancer genes are used as probes todetermine the number of copies of the cancer gene in the genome. Thecancer genes are used as probes to determine the chromosomallocalization of the cancer genes. Information such as chromosomallocalization finds use in providing a diagnosis or prognosis inparticular when chromosomal abnormalities such as translocations, andthe like are identified in the cancer gene locus.

XIV.) Kits/Articles of Manufacture

For use in the laboratory, prognostic, prophylactic, diagnostic andtherapeutic applications described herein, kits are within the scope ofthe invention. Such kits can comprise a carrier, package, or containerthat is compartmentalized to receive one or more containers such asvials, tubes, and the like, each of the container(s) comprising one ofthe separate elements to be used in the method, along with a label orinsert comprising instructions for use, such as a use described herein.For example, the container(s) can comprise a probe that is or can bedetectably labeled. Such probe can be an antibody or polynucleotidespecific for a protein or a gene or message of the invention,respectively. Where the method utilizes nucleic acid hybridization todetect the target nucleic acid, the kit can also have containerscontaining nucleotide(s) for amplification of the target nucleic acidsequence. Kits can comprise a container comprising a reporter, such as abiotin-binding protein, such as avidin or streptavidin, bound to areporter molecule, such as an enzymatic, fluorescent, or radioisotopelabel; such a reporter can be used with, e.g., a nucleic acid orantibody. The kit can include all or part of the amino acid sequences inFIG. 2 or FIG. 3 or analogs thereof, or a nucleic acid molecule thatencodes such amino acid sequences.

The kit of the invention will typically comprise the container describedabove and one or more other containers associated therewith thatcomprise materials desirable from a commercial and user standpoint,including buffers, diluents, filters, needles, syringes; carrier,package, container, vial and/or tube labels listing contents and/orinstructions for use, and package inserts with instructions for use.

A label can be present on or with the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, such as a prognostic, prophylactic, diagnostic orlaboratory application, and can also indicate directions for either invivo or in vitro use, such as those described herein. Directions and orother information can also be included on an insert(s) or label(s) whichis included with or on the kit. The label can be on or associated withthe container. A label a can be on a container when letters, numbers orother characters forming the label are molded or etched into thecontainer itself; a label can be associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. The label can indicate that the compositionis used for diagnosing, treating, prophylaxing or prognosing acondition, such as a neoplasia of a tissue set forth in Table I.

The terms “kit” and “article of manufacture” can be used as synonyms.

In another embodiment of the invention, an article(s) of manufacturecontaining compositions, such as amino acid sequence(s), smallmolecule(s), nucleic acid sequence(s), and/or antibody(s), e.g.,materials useful for the diagnosis, prognosis, prophylaxis and/ortreatment of neoplasias of tissues such as those set forth in Table I isprovided. The article of manufacture typically comprises at least onecontainer and at least one label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers can beformed from a variety of materials such as glass, metal or plastic. Thecontainer can hold amino acid sequence(s), small molecule(s), nucleicacid sequence(s), cell population(s) and/or antibody(s). In oneembodiment, the container holds a polynucleotide for use in examiningthe mRNA expression profile of a cell, together with reagents used forthis purpose. In another embodiment a container comprises an antibody,binding fragment thereof or specific binding protein for use inevaluating protein expression of 158P3D2 in cells and tissues, or forrelevant laboratory, prognostic, diagnostic, prophylactic andtherapeutic purposes; indications and/or directions for such uses can beincluded on or with such container, as can reagents and othercompositions or tools used for these purposes. In another embodiment, acontainer comprises materials for eliciting a cellular or humoral immuneresponse, together with associated indications and/or directions. Inanother embodiment, a container comprises materials for adoptiveimmunotherapy, such as cytotoxic T cells (CTL) or helper T cells (HTL),together with associated indications and/or directions; reagents andother compositions or tools used for such purpose can also be included.

The container can alternatively hold a composition that is effective fortreating, diagnosis, prognosing or prophylaxing a condition and can havea sterile access port (for example the container can be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agents in the composition can be anantibody capable of specifically binding 158P3D2 and modulating thefunction of 158P3D2.

The article of manufacture can further comprise a second containercomprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and/or dextrose solution.It can further include other materials desirable from a commercial anduser standpoint, including other buffers, diluents, filters, stirrers,needles, syringes, and/or package inserts with indications and/orinstructions for use.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which is intended tolimit the scope of the invention.

Example 1 SSH-Generated Isolation of a cDNA Fragment of the 158P3D2 Gene

To isolate genes that are over-expressed in bladder cancer we used theSuppression Subtractive Hybridization (SSH) procedure using cDNA derivedfrom bladder cancer tissues, including invasive transitional cellcarcinoma. The 158P3D2 SSH cDNA sequence was derived from a bladdercancer pool minus normal bladder cDNA subtraction. Included in thedriver were also cDNAs derived from 9 other normal tissues. The 158P3D2cDNA was identified as highly expressed in the bladder cancer tissuepool, with lower expression seen in a restricted set of normal tissues.

The SSH DNA sequence of 312 bp (FIG. 1) shows identity to the fer-1-like4 (C. elegans) (FER1L4) mRNA. A 158P3D2 cDNA clone 158P3D2-BCP1 of 1994bp was isolated from bladder cancer cDNA, revealing an ORF of 328 aminoacids (FIG. 2, FIG. 3).

Amino acid sequence analysis of 158P3D2 reveals 100% identity over 328amino acid region to dJ477O4.1.1, a novel protein similar to otoferlinand dysferlin, isoform 1 protein (GenBank Accession CAB89410.1).

The 158P3D2 protein has a transmembrane domain of 23 residues betweenamino acids 292-313 predicted by the SOSUI Signal program.

Materials and Methods

Human Tissues:

The patient cancer and normal tissues were purchased from differentsources such as the NDRI (Philadelphia, Pa.). mRNA for some of thenormal tissues were purchased from Clontech, Palo Alto, Calif.

RNA Isolation:

Tissues were homogenized in Trizol reagent (Life Technologies, GibcoBRL) using 10 ml/g tissue isolate total RNA. Poly A RNA was purifiedfrom total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Totaland mRNA were quantified by spectrophotometric analysis (O.D. 260/280nm) and analyzed by gel electrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used. DPNCDN (cDNAsynthesis primer): (SEQ ID NO: 67) 5′TTTTGATCAAGCTT₃₀3′ Adaptor 1: (SEQID NO: 68) 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NO:69) 3′GGCCCGTCCTAG5′ Adaptor 2: (SEQ ID NO: 70)5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 71)3′CGGCTCCTAG5′ PCR primer 1: (SEQ ID NO: 72) 5′CTAATACGACTCACTATAGGGC3′Nested primer (NP)1: (SEQ ID NO: 73) 5′TCGAGCGGCCGCCCGGGCAGGA3′ Nestedprimer (NP)2: (SEQ ID NO: 74) 5′AGCGTGGTCGCGGCCGAGGA3′

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes that may be differentially expressed in bladdercancer. The SSH reaction utilized cDNA from bladder cancer and normaltissues.

The gene 158P3D2 sequence was derived from a bladder cancer pool minusnormal bladder cDNA subtraction. The SSH DNA sequence (FIG. 1) wasidentified.

The cDNA derived from of pool of normal bladder tissues was used as thesource of the “driver” cDNA, while the cDNA from a pool of bladdercancer tissues was used as the source of the “tester” cDNA. Doublestranded cDNAs corresponding to tester and driver cDNAs were synthesizedfrom 2 μg of poly(A)⁺ RNA isolated from the relevant xenograft tissue,as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and1 ng of oligonucleotide DPNCDN as primer. First- and second-strandsynthesis were carried out as described in the Kit's user manualprotocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). Theresulting cDNA was digested with Dpn II for 3 hrs at 37° C. DigestedcDNA was extracted with phenol/chloroform (1:1) and ethanolprecipitated.

Driver cDNA was generated by combining in a 1:1 ratio Dpn II digestedcDNA from the relevant tissue source (see above) with a mix of digestedcDNAs derived from the nine normal tissues: stomach, skeletal muscle,lung, brain, liver, kidney, pancreas, small intestine, and heart.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA fromthe relevant tissue source (see above) (400 ng) in 5 μl of water. Thediluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 andAdaptor 2 (10 μM), in separate ligation reactions, in a total volume of10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH).Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C.for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR productswere analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight.

To identify inserts, PCR amplification was performed on 1 ml ofbacterial culture using the conditions of PCR1 and NP1 and NP2 asprimers. PCR products were analyzed using 2% agarose gelelectrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs can be generated from 1 μg of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system.The manufacturer's protocol was used which included an incubation for 50min at 42° C. with reverse transcriptase followed by RNAse H treatmentat 37° C. for 20 min. After completing the reaction, the volume can beincreased to 200 μl with water prior to normalization. First strandcDNAs from 16 different normal human tissues can be obtained fromClontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO:75) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 76) to amplifyβ-actin. First strand cDNA (5 μl) were amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1× Klentaq DNApolymerase (Clontech). Five μl of the PCR reaction can be removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: Initial denaturation can be at 94° C. for 15 sec, followedby a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C.for 5 sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 b.p.β-actin bands from multiple tissues were compared by visual inspection.Dilution factors for the first strand cDNAs were calculated to result inequal β-actin band intensities in all tissues after 22 cycles of PCR.Three rounds of normalization can be required to achieve equal bandintensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 158P3D2 gene, 5 μl of normalizedfirst strand cDNA were analyzed by PCR using 26, and 30 cycles ofamplification. Semi-quantitative expression analysis can be achieved bycomparing the PCR products at cycle numbers that give light bandintensities. The primers used for RT-PCR were designed using the 158P3D2SSH sequence and are listed below: 158P3D2.1 5′ CATCTATGTGAAGAGCTGGGTGAA3′ (SEQ ID NO: 77) 158P3D2.2 5′ AGGTAGTCAAAGCGGAACACAAAG 3′ (SEQ ID NO:78)

Additional primers were also designed to test for expression of thedifferent splice variants and these are listed below: Primer NameSequence 158P3D2 ex. 17 - R GTCCTCCCAGCAACTCCACACA (SEQ ID NO: 79)158P3D2 ex. 26 - F TGTCCCTTCCACCCAACGTGTGC (SEQ ID NO: 80) 158P3D2 ex.28 - R TCCTCCATCTCTCCTTCCTCCTCAG (SEQ ID NO: 81) 158P3D2 ex. 9 - FCAGAAACTGGTGGGAGTCAACA (SEQ ID NO: 82) 158P3D2 ex. 1 - FATGGCTCTGACGGTAAGCGTGC (SEQ ID NO: 83) 158P3D2 ex. 10 - FATAGGCACCTTCAGGATGGACC (SEQ ID NO: 84) 158P3D2 ex. 10 - RTCCATCCTGAAGGTGCCTATCC (SEQ ID NO: 85) 158P3D2 ex. 16 - FCAGAGGAGGAGAAAGAGGAGG (SEQ ID NO: 86) 158P3D2 ex. 16 - RTCCTCTTTCTCCTCCTCTGG (SEQ ID NO: 87) 158P3D2 ex. 21 - FAGATCCAGAGTCTAATGCTCACG (SEQ ID NO: 88) 158P3D2 ex. 21 - RCGTGAGCATTAGACTCTGGATC (SEQ ID NO: 89) 158P3D2 ex. 27 - FAAGGTGTGGAGTCTGAGGTC (SEQ ID NO: 90) 158P3D2 ex. 27 - RACCTCAGACTCCACACCTTGC (SEQ ID NO: 91) 158P3D2 ex. 34 - RACTCTGACCAGGAGCTTGATG (SEQ ID NO: 92) 158P3D2 ex. 40 - FACACGGAGGATGTGGTTCTGG (SEQ ID NO: 93) 158P3D2 ex. 43 - FTTGAGCTGCTGACTGTGGAGGAG (SEQ ID NO: 94) 158P3D2 ex. 43 - RTCCTCCACAGTCAGCAGCTC (SEQ ID NO: 95) 158P3D2 ex. 44 - RTGAGTGTCCAAGGTCAGCGAG (SEQ ID NO: 96) 158P3D2 ex. 7 - FAGAGAATGAGCTGGAGCTTGAGC (SEQ ID NO: 97) 158P3D2 ex. 7 - RTCAAGCTCCAGCTCATTCTCTTC (SEQ ID NO: 98) AGS-25 long RT PCR-3′TAACACCAGAAAGTTCCACGTCAG (SEQ ID NO: 99) AGS-25 long RT PCR-5′TGACGGTCGCCGTATTTGATC (SEQ ID NO: 100) AGS-25 short RT PCR-3′GATTGGCTGCCGAGGCTTGA (SEQ ID NO: 101) AGS-25 short RT PCR-5′TGACGGTCGCCGTATTTGATC (SEQ ID NO: 102)

A typical RT-PCR expression analysis is shown in FIG. 14. RT-PCRexpression analysis was performed on first strand cDNAs generated usingpools of tissues from multiple samples. The cDNAs were shown to benormalized using beta-actin PCR. Results show strong expression of158P3D2 in bladder cancer pool, kidney cancer pool and cancer metastasispool. Expression of 158P3D2 is also detected in colon cancer pool, lungcancer pool, ovary cancer pool, breast cancer pool, pancreas cancer pooland prostate metastases to lymph node, and vital pool 2, but not vitalpool 1.

Example 2 Full Length Cloning of 158P3D2

The 158P3D2 SSH cDNA sequence was derived from a bladder cancer poolminus normal bladder cDNA subtraction. The SSH cDNA sequence (FIG. 1)was designated 158P3D2. The full-length cDNA clone 158P3D2 v.1 clone158P3D2-BCP1 and 158P3D2-BCP2 (FIG. 2) were cloned from bladder cancerpool cDNA.

Additional 158P3D2 splice and SNP variants have been identified andthese are listed in FIG. 2 and FIG. 3.

Example 3 Chromosomal Mapping of 158P3D2

Chromosomal localization can implicate genes in disease pathogenesis.Several chromosome mapping approaches are available includingfluorescent in situ hybridization (FISH), human/hamster radiation hybrid(RH) panels (Walter et al., 1994; Nature Genetics 7:22; ResearchGenetics, Huntsville Ala.), human-rodent somatic cell hybrid panels suchas is available from the Coriell Institute (Camden, N.J.), and genomicviewers utilizing BLAST homologies to sequenced and mapped genomicclones (NCBI, Bethesda, Md.).

158P3D2 maps to chromosome 8, using 158P3D2 sequence and the NCBI BLASTtool located on the World Wide Web at:.(ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs).

Example 4 Expression Analysis of 158P3D2 in Normal Tissues and PatientSpecimens

Expression analysis by RT-PCR demonstrated that 158P3D2 is stronglyexpressed in multiple cancer patient specimens, but unrestricted normaltissues (FIG. 14). First strand cDNA was prepared from a panel of 13normal tissues (brain, heart, kidney, liver, lung, spleen, skeletalmuscle, testis, pancreas, colon, stomach) and pools of 4-7 patients fromthe following cancer indications: bladder, kidney, colon, lung,pancreas, stomach, ovary, breast, multiple cancer metastasis, cervix,lymphoma as well as from a pool of patient-derived xenografts (prostatecancer, bladder cancer and kidney cancer). Normalization was performedby PCR using primers to actin and GAPDH. Semi-quantitative PCR, usingprimers to 158P3D2, was performed at 26 and 30 cycles of amplification.Samples were run on an agarose gel, and PCR products were quantitatedusing the AlphaImager software. Results show strong expression of158P3D2 in cancers of the bladder, kidney, colon, lung, pancreas,stomach, ovary, breast, cervix, and lymphoma. Strong expression was alsoobserved in the cancer metastasis pool. Low expression was detected inall normal tissues tested except in normal stomach.

Expression of 158P3D2 in bladder cancer patient specimens and humannormal tissues is shown in FIG. 15. First strand cDNA was prepared fromnormal bladder, bladder cancer cell lines (UM-UC-3, TCCSUP, J82) and apanel of bladder cancer patient specimens. Normalization was performedby PCR using primers to actin and GAPDH. Expression level was recordedas no expression (no signal detected), low (signal detected at 30×),medium (signal detected at 26×), high (strong signal at 26×). Resultsshow expression of 158P3D2 in the majority of bladder cancer patientspecimens tested. Very low expression was detected in normal tissues,but no expression was seen in the cell lines tested.

Northern blot analysis of 158P3D2 in bladder specimens is shown in FIG.16. RNA was extracted from normal bladder, bladder cancer cell lines(UM-UC-3, J82, SCaBER), bladder cancer patient tumors (T) and theirnormal adjacent tissues (NAT). Northern blot with 10 μg of total RNAwere probed with the 158P3D2 sequence. Size standards in kilobases areon the side. Results show strong expression of 158P3D2 in tumor tissues,but not in normal, nor NAT tissues.

FIG. 17 shows 158P3D2 expression in lung cancer patient specimens. Firststrand cDNA was prepared from normal lung, cancer cell line A427 and apanel of lung cancer patient specimens. Normalization was performed byPCR using primers to actin and GAPDH. Semi-quantitative PCR, usingprimers to 158P3D2, was performed at 26 and 30 cycles of amplification.Expression level was recorded as no expression (no signal detected), low(signal detected at 30×), medium (signal detected at 26×), high (strongsignal at 26×). 158P3D2 is expressed at varying levels in 35/39 (90%) oflung cancer specimens, but not in all 3 normal lung tissues tested.

Northern blot analysis of 158P3D2 expression in lung cancer patientspecimens is shown in FIG. 18. RNA was extracted from normal lung, A427lung cancer cell line, and a panel of lung cancer patient specimens.Northern blot with 10 μg of total RNA were probed with the 158P3D2sequence. Size standards in kilobases are on the side. Results showstrong expression of 158P3D2 in tumor specimens but not in normaltissues.

FIG. 19 shows 158P3D2 expression in cancer metastasis patient specimens.First strand cDNA was prepared from normal colon, kidney, liver, lung,pancreas, stomach and from a panel of cancer metastasis patientspecimens. Normalization was performed by PCR using primers to actin andGAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at26 and 30 cycles of amplification. Expression level was recorded as noexpression (no signal detected), low (signal detected at 30×), medium(signal detected at 26×), high (strong signal at 26×). Results showexpression of 158P3D2 in the majority of patient cancer metastasisspecimens tested but not in normal tissues.

FIG. 20 shows 158P3D2 expression in cervical cancer patient specimens.First strand cDNA was prepared from normal cervix, cervical cancer cellline HeLa, and a panel of cervical cancer patient specimens.Normalization was performed by PCR using primers to actin and GAPDH.Expression level was recorded as no expression (no signal detected), low(signal detected at 30×), medium (signal detected at 26×), high (strongsignal at 26×). Results show expression of 158P3D2 in all 14 cervicalcancer patient specimens tested. No expression was detected in normalcervix or in the cell line tested.

Northern blot analysis of 158P3D2 expression in cervical cancer patientspecimens is shown in FIG. 21. RNA was extracted from normal cervix,cervical cancer cell line HeLa, and a panel of cervical cancer patientspecimens. Northern blot with 10 μg of total RNA were probed with the158P3D2 sequence. Size standards in kilobases are on the side. Resultsshow strong expression of 158P3D2 in tumor tissues, but not in normalcervix nor in the cell line.

FIG. 22 shows 158P3D2 expression in kidney cancer patient specimens.First strand cDNA was prepared from normal kidney, kidney cancer celllines (769-P, A-498, CAKI-1), and a panel of kidney cancer patientspecimens. Normalization was performed by PCR using primers to actin andGAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at26 and 30 cycles of amplification. Expression level was recorded as noexpression (no signal detected), low (signal detected at 30×), medium(signal detected at 26×), high (strong signal at 26×). 158P3D2 isexpressed at varying levels in the majority of kidney cancer patientspecimens, but not in all 3 normal kidney tissues tested. Low expressionwas detected in 2 of 3 cell lines tested.

FIG. 23 shows 158P3D2 expression in kidney cancer patient specimens bynorthern blotting. RNA was extracted from normal kidney and a panel ofkidney cancer patient specimens. Northern blot with 10 μg of total RNAwere probed with the 158P3D2 sequence. Size standards in kilobases areon the side. Results show strong expression of 158P3D2 in tumorspecimens but not in the normal tissue.

FIG. 24 shows 158P3D2 expression in stomach cancer patient specimens.First strand cDNA was prepared from normal stomach, and a panel ofstomach cancer patient specimens. Normalization was performed by PCRusing primers to actin and GAPDH. Semi-quantitative PCR, using primersto 158P3D2, was performed at 26 and 30 cycles of amplification.Expression level was recorded as no expression (no signal detected), low(signal detected at 30×), medium (signal detected at 26×), high (strongsignal at 26×). 158P3D2 is expressed at varying levels in the majorityof stomach cancer patient specimens. Weak expression was detected in the2 normal stomach, and only in 1 of the 2 NAT tissues tested.

FIG. 25 shows 158P3D2 expression in stomach cancer patient specimens bynorthern blotting. RNA was extracted from normal stomach and a panel ofstomach cancer patient specimens. Northern blot with 10 μg of total RNAwere probed with the 158P3D2 sequence. Size standards in kilobases areon the side. Results show strong expression of 158P3D2 in tumorspecimens but not in the normal tissue.

FIG. 26 shows 158P3D2 expression in colon cancer patient specimens.First strand cDNA was prepared from normal colon, colon cancer celllines (LoVo, CaCO-2, SK CO 1, Colo 205, T284), and a panel of coloncancer patient specimens. Normalization was performed by PCR usingprimers to actin and GAPDH. Semi-quantitative PCR, using primers to158P3D2, was performed at 26 and 30 cycles of amplification. Expressionlevel was recorded as no expression (no signal detected), low (signaldetected at 30×s), medium (signal detected at 26×), high (strong signalat 26×). 158P3D2 is expressed at varying levels in the majority of coloncancer patient specimens. But it was weakly expressed in just 2 of 3normal tissues, and 3 of 5 cell lines tested.

FIG. 27 shows 158P3D2 expression in uterus cancer patient specimens.First strand cDNA was prepared from normal uterus and a panel of uteruscancer patient specimens. Normalization was performed by PCR usingprimers to actin and GAPDH. Semi-quantitative PCR, using primers to158P3D2, was performed at 26 and 30 cycles of amplification. Expressionlevel was recorded as no expression (no signal detected), low (signaldetected at 30×), medium (signal detected at 26×), high (strong signalat 26×). Results show 158P3D2 is expressed at varying levels in themajority of uterus cancer patient specimens, but not in normal uterus.

FIG. 28 shows 158P3D2 expression in breast cancer patient specimens.First strand cDNA was prepared from normal breast, breast cancer celllines (MD-MBA-435S, DU4475, MCF-7, CAMA-1, MCF10A), and a panel ofbreast cancer patient specimens. Normalization was performed by PCRusing primers to actin and GAPDH. Semi-quantitative PCR, using primersto 158P3D2, was performed at 26 and 30 cycles of amplification.Expression level was recorded as no expression (no signal detected), low(signal detected at 30×), medium (signal detected at 26×), high (strongsignal at 26×). Results show 158P3D2 is expressed at varying levels inthe majority of breast cancer patient specimens. But it was weaklyexpressed in just 2 of 3 normal tissues, and 2 of 5 cell lines tested.

The restricted expression of 158P3D2 in normal tissues and theexpression detected in bladder cancer, kidney cancer, colon cancer, lungcancer, pancreas cancer, stomach cancer, ovary cancer, breast cancer,uterus cancer, cervical cancer and lymphoma suggest that 158P3D2 is apotential therapeutic target and a diagnostic marker for the treatmentof human cancers.

Example 5 Transcript Variants of 158P3D2

Transcript variants are variants of mature mRNA from the same gene whicharise by alternative transcription or alternative splicing. Alternativetranscripts are transcripts from the same gene but start transcriptionat different points. Splice variants are mRNA variants spliceddifferently from the same transcript. In eukaryotes, when a multi-exongene is transcribed from genomic DNA, the initial RNA is spliced toproduce functional mRNA, which has only exons and is used fortranslation into an amino acid sequence. Accordingly, a given gene canhave zero to many alternative transcripts and each transcript can havezero to many splice variants. Each transcript variant has a unique exonmakeup, and can have different coding and/or non-coding (5′ or 3′ end)portions, from the original transcript. Transcript variants can code forsimilar or different proteins with the same or a similar function or canencode proteins with different functions, and can be expressed in thesame tissue at the same time, or in different tissues at the same time,or in the same tissue at different times, or in different tissues atdifferent times. Proteins encoded by transcript variants can havesimilar or different cellular or extracellular localizations, e.g.,secreted versus intracellular.

Transcript variants are identified by a variety of art-accepted methods.For example, alternative transcripts and splice variants are identifiedby full-length cloning experiment, or by use of full-length transcriptand EST sequences. First, all human ESTs were grouped into clusterswhich show direct or indirect identity with each other. Second, ESTs inthe same cluster were further grouped into sub-clusters and assembledinto a consensus sequence. The original gene sequence is compared to theconsensus sequence(s) or other full-length sequences. Each consensussequence is a potential splice variant for that gene. Even when avariant is identified that is not a full-length clone, that portion ofthe variant is very useful for antigen generation and for furthercloning of the full-length splice variant, using techniques known in theart.

Moreover, computer programs are available in the art that identifytranscript variants based on genomic sequences. Genomic-based transcriptvariant identification programs include FgenesH (A. Salamov and V.Solovyev,. “Ab initio gene finding in Drosophila genomic DNA,” GenomeResearch. 2000 April; 10(4):516-22); Grail (URLcompbio.oml.gov/Grail-bin/EmptyGrailForm) and GenScan (URLgenes.mit.edu/GENSCAN.html). For a general discussion of splice variantidentification protocols see., e.g., Southan, C., A genomic perspectiveon human proteases, FEBS Lett. 2001 Jun. 8; 498(2-3):214-8; de Souza, S.J., et al., Identification of human chromosome 22 transcribed sequenceswith ORF expressed sequence tags, Proc. Natl. Acad Sci USA. 2000 Nov. 7;97(23):12690-3.

To further confirm the parameters of a transcript variant, a variety oftechniques are available in the art, such as full-length cloning,proteomic validation, PCR-based validation, and 5′ RACE validation, etc.(see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin bankspeninsula: a new termination variant characterized by electrospray massspectrometry, Biochem Biophys Acta. 1999 Aug. 17;1433(1-2):321-6;Ferranti P, et al., Differential splicing of pre-messenger RNA producesmultiple forms of mature caprine alpha(s1)-casein, Eur J. Biochem. 1997Oct. 1; 249(1):1-7. For PCR-based Validation: Wellmann S, et al.,Specific reverse transcription-PCR quantification of vascularendothelial growth factor (VEGF) splice variants by LightCyclertechnology, Clin Chem. 2001 April; 47(4):654-60; Jia, H. P., et al.,Discovery of new human beta-defensins using a genomics-based approach,Gene. 2001 Jan. 24; 263(1-2):211-8. For PCR-based and 5′ RACEValidation: Brigle, K. E., et al., Organization of the murine reducedfolate carrier gene and identification of variant splice forms, BiochemBiophys Acta. 1997 Aug. 7; 1353(2): 191-8).

It is known in the art that genomic regions are modulated in cancers.When the genomic region to which a gene maps is modulated in aparticular cancer, the alternative transcripts or splice variants of thegene are modulated as well. Disclosed herein is that 158P3D2 has aparticular expression profile related to cancer. Alternative transcriptsand splice variants of 158P3D2 may also be involved in cancers in thesame or different tissues, thus serving as tumor-associatedmarkers/antigens.

Using the full-length gene and EST sequences, six transcript variantswere identified, designated as 158P3D2 v.2, v.14 through v.18. Theboundaries of the exon in the original transcript, 158P3D2 v.1 wereshown in Table LI. Exon compositions of the variants are shown in FIG.10. Each different combination of exons in spatial order, e.g. exon 1 ofv.2 and exons 3, 4, 5 and 6 of v.1, is a potential splice variant.

Tables LII(a)-(f) through LV(a)-(f) are set forth on avariant-by-variant bases. Tables LII(a)-(f) show nucleotide sequence ofthe transcript variants. Tables LIII(a)-(f) show the alignment of therespective transcript variant with nucleic acid sequence of 158P3D2 v.1.Tables LIV(a)-(f) lay out amino acid translation of the transcriptvariants for the identified reading frame orientation. Tables LV(a)-(f)displays alignments of the amino acid sequence encoded by the splicevariant with that of 158P3D2 v.1.

Example 6 Single Nucleotide Polymorphisms of 158P3D2

A Single Nucleotide Polymorphism (SNP) is a single base pair variationin a nucleotide sequence at a specific location. At any given point ofthe genome, there are four possible nucleotide base pairs: A/T, C/G, G/Cand T/A. Genotype refers to the specific base pair sequence of one ormore locations in the genome of an individual. Haplotype refers to thebase pair sequence of more than one location on the same DNA molecule(or the same chromosome in higher organisms), often in the context ofone gene or in the context of several tightly linked genes. SNP thatoccurs on a cDNA is called cSNP. This cSNP may change amino acids of theprotein encoded by the gene and thus change the functions of theprotein. Some SNP cause inherited diseases; others contribute toquantitative variations in phenotype and reactions to environmentalfactors including diet and drugs among individuals. Therefore, SNPand/or combinations of alleles (called haplotypes) have manyapplications, including diagnosis of inherited diseases, determinationof drug reactions and dosage, identification of genes responsible fordiseases, and analysis of the genetic relationship between individuals(P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect humantraits,” Curr. Opin. Neurobiol. 2001 October; 11(5):637-641; M.Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drugreactions,” Trends Pharmacol. Sci. 2001 June; 22(6):298-305; J. H.Riley, C. J. Allan, E. Lai and A. Roses, “The use of single nucleotidepolymorphisms in the isolation of common disease genes,”Pharmacogenomics. 2000 February; 1(1):39-47; R. Judson, J. C. Stephensand A. Windemuth, “The predictive power of haplotypes in clinicalresponse,” Pharmacogenomics. 2000 February; 1(1):15-26).

SNP are identified by a variety of art-accepted methods (P. Bean, “Thepromising voyage of SNP target discovery,” Am. Clin. Lab. 2001October-November; 20(9): 18-20; K. M. Weiss, “In search of humanvariation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enablinglarge-scale pharmacogenetic studies by high-throughput mutationdetection and genotyping technologies,” Clin. Chem. 2001 February;47(2): 164-172). For example, SNP can be identified by sequencing DNAfragments that show polymorphism by gel-based methods such asrestriction fragment length polymorphism (RFLP) and denaturing gradientgel electrophoresis (DGGE). They can also be discovered by directsequencing of DNA samples pooled from different individuals or bycomparing sequences from different DNA samples. With the rapidaccumulation of sequence data in public and private databases, one candiscover SNP by comparing sequences using computer programs (Z. Gu, L.Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting incyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNP can be verified andgenotype or haplotype of an individual can be determined by a variety ofmethods including direct sequencing and high throughput microarrays (P.Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu.Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K.Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft,“High-throughput SNP genotyping with the Masscode system,” Mol. Diagn.2000 December; 5(4):329-340).

Using the methods described above, twelve SNP were identified in theoriginal transcript, 158P3D2 v.1, at positions 1155 (T/C), 1152 (G/A),960 (G/T) and 1236 (G/-), 519 (A/G), 440 (T/A), 971 (T/C), 150 (C/G),1022 (C/A), 1148 (G/A), 1691 (G/T) and 1692 (A/G). The transcripts orproteins with alternative allele were designated as variant 158P3D2 v.3through v.13, respectively. FIG. 12 shows the schematic alignment of theSNP variants. FIG. 11 shows the schematic alignment of protein variants,corresponding to nucleotide variants. Nucleotide variants that code forthe same amino acid sequence as v.1 are not shown in FIG. 11. Thesealleles of the SNP, though shown separately here, can occur in differentcombinations (haplotypes) and in any one of the transcript variants(such as 158P3D2 v.17) that contains the site of the SNP.

Example 7 Production of Recombinant 158P3D2 in Prokaryotic Systems

To express recombinant 158P3D2 and 158P3D2 variants in prokaryoticcells, the full or partial length 158P3D2 and 158P3D2 variant cDNAsequences are cloned into any one of a variety of expression vectorsknown in the art. One or more of the following regions of 158P3D2variants are expressed: the full length sequence presented in FIGS. 2and 3, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from158P3D2, variants, or analogs thereof.

A. In Vitro Transcription and Translation Constructs

pCRII: To generate 158P3D2 sense and anti-sense RNA probes for RNA insitu investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) aregenerated encoding either all or fragments of the 158P3D2 cDNA. ThepCRII vector has Sp6 and T7 promoters flanking the insert to drive thetranscription of 158P3D2 RNA for use as probes in RNA in situhybridization experiments. These probes are used to analyze the cell andtissue expression of 158P3D2 at the RNA level. Transcribed 158P3D2 RNArepresenting the cDNA amino acid coding region of the 158P3D2 gene isused during in vitro translation systems such as the TnT™ CoupledReticulolysate System (Promega, Corp., Madison, Wis.) to synthesize158P3D2 protein.

B. Bacterial Constructs

pGEX Constructs: To generate recombinant 158P3D2 proteins in bacteriathat are fused to the Glutathione S-transferase (GST) protein, all orparts of the 158P3D2 cDNA protein coding sequence are cloned into thepGEX family of GST-fusion vectors (Amersham Pharmacia Biotech,Piscataway, N.J.). These constructs allow controlled expression ofrecombinant 158P3D2 protein sequences with GST fused at theamino-terminus and a six histidine epitope (6×His) at thecarboxyl-terminus. The GST and 6×His tags permit purification of therecombinant fusion protein from induced bacteria with the appropriateaffinity matrix and allow recognition of the fusion protein withanti-GST and anti-His antibodies. The 6×His tag is generated by adding 6histidine codons to the cloning primer at the 3′ end, e.g., of the openreading frame (ORF). A proteolytic cleavage site, such as thePreScission™ recognition site in pGEX-6P-1, may be employed such that itpermits cleavage of the GST tag from 158P3D2-related protein. Theampicillin resistance gene and pBR322 origin permits selection andmaintenance of the pGEX plasmids in E. coli.

pMAL Constructs: To generate, in bacteria, recombinant 158P3D2 proteinsthat are fused to maltose-binding protein (MBP), all or parts of the158P3D2 cDNA protein coding sequence are fused to the MBP gene bycloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs,Beverly, Mass.). These constructs allow controlled expression ofrecombinant 158P3D2 protein sequences with MBP fused at theamino-terminus and a 6×His epitope tag at the carboxyl-terminus. The MBPand 6×His tags permit purification of the recombinant protein frominduced bacteria with the appropriate affinity matrix and allowrecognition of the fusion protein with anti-MBP and anti-His antibodies.The 6×His epitope tag is generated by adding 6 histidine codons to the3′ cloning primer. A Factor Xa recognition site permits cleavage of thepMAL tag from 158P3D2. The pMAL-c2X and pMAL-p2X vectors are optimizedto express the recombinant protein in the cytoplasm or periplasmrespectively. Periplasm expression enhances folding of proteins withdisulfide bonds.

pET Constructs: To express 158P3D2 in bacterial cells, all or parts ofthe 158P3D2 cDNA protein coding sequence are cloned into the pET familyof vectors (Novagen, Madison, Wis.). These vectors allow tightlycontrolled expression of recombinant 158P3D2 protein in bacteria withand without fusion to proteins that enhance solubility, such as NusA andthioredoxin (Trx), and epitope tags, such as 6×His and S-Tag™ that aidpurification and detection of the recombinant protein. For example,constructs are made utilizing pET NusA fusion system 43.1 such thatregions of the 158P3D2 protein are expressed as amino-terminal fusionsto NusA. The cDNA encoding amino acids 155-290 and amino acids 260-328of 158P3D2 each were cloned into the pET-21b vector. The recombinantproteins can be used to generate rabbit polyclonal antibodies.

C. Yeast Constructs:

pESC Constructs: To express 158P3D2 in the yeast species Saccharomycescerevisiae for generation of recombinant protein and functional studies,all or parts of the 158P3D2 cDNA protein coding sequence are cloned intothe pESC family of vectors each of which contain 1 of 4 selectablemarkers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.).These vectors allow controlled expression from the same plasmid of up to2 different genes or cloned sequences containing either Flag or Mycepitope tags in the same yeast cell. This system is useful to confirmprotein-protein interactions of 158P3D2. In addition, expression inyeast yields similar post-translational modifications, such asglycosylations and phosphorylations, that are found when expressed ineukaryotic cells.

pESP Constructs: To express 158P3D2 in the yeast species Saccharomycespombe, all or parts of the 158P3D2 cDNA protein coding sequence arecloned into the pESP family of vectors. These vectors allow controlledhigh level of expression of a 158P3D2 protein sequence that is fused ateither the amino terminus or at the carboxyl terminus to GST which aidspurification of the recombinant protein. A Flag™ epitope tag allowsdetection of the recombinant protein with anti-Flag™ antibody.

Example 8 Production of Recombinant 158P3D2 in Higher Eukaryotic Systems

A. Mammalian Constructs:

To express recombinant 158P3D2 in eukaryotic cells, the full or partiallength 158P3D2 cDNA sequences, or variants thereof, can be cloned intoany one of a variety of expression vectors known in the art. One or moreof the following regions of 158P3D2 are expressed in these constructs,amino acids 1 to 328, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous aminoacids from 158P3D2 v.1, v.3, v.4, v.10, v.12 and v.13; amino acids 1 to236, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from158P3D2v.2A; amino acids 1 to 181, or any 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morecontiguous amino acids from 158P3D2 v.2B or v.5B; amino acids 1 to 178,or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more contiguous amino acids from 158P3D2 v.5A;amino acids 1 to 2036, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous aminoacids from 158P3D2 v.17; amino acids 1 to 1990, or any 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30or more contiguous amino acids from 158P3D2v.16; amino acids 1 to 1145,or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30 or more contiguous amino acids from 158P3D2 v.15;amino acids 1 to 1393, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous aminoacids from 158P3D2 v.14; amino acids 1 to 610, or any 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30or more contiguous amino acids from 158P3D2v.18; or any 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30 or more contiguous amino acids from 282P1G3 variants, or analogsthereof.

The constructs can be transfected into any one of a wide variety ofmammalian cells such as 293T cells. Transfected 293T cell lysates can beprobed with the anti-158P3D2 polyclonal serum, described herein.

-   -   pcDNA4/HisMax Constructs: To express 158P3D2 in mammalian cells,        a 158P3D2 ORF, or portions thereof, of 158P3D2 are cloned into        pcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Protein        expression is driven from the cytomegalovirus (CMV) promoter and        the SP16 translational enhancer. The recombinant protein has        Xpress™ and six histidine (6×His) epitopes fused to the        amino-terminus. The pcDNA4/HisMax vector also contains the        bovine growth hormone (BGH) polyadenylation signal and        transcription termination sequence to enhance mRNA stability        along with the SV40 origin for episomal replication and simple        vector rescue in cell lines expressing the large T antigen. The        Zeocin resistance gene allows for selection of mammalian cells        expressing the protein and the ampicillin resistance gene and        ColE1 origin permits selection and maintenance of the plasmid        in E. coli.

pcDNA3.1/MycHis Constructs: To express 158P3D2 in mammalian cells, a158P3D2 ORF, or portions thereof, of 158P3D2 with a consensus Kozaktranslation initiation site was cloned into pcDNA3.1/MycHis Version A(Invitrogen, Carlsbad, Calif.). Protein expression is driven from thecytomegalovirus (CMV) promoter. The recombinant proteins have the mycepitope and 6×His epitope fused to the carboxyl-terminus. ThepcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability, along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheNeomycin resistance gene can be used, as it allows for selection ofmammalian cells expressing the protein and the ampicillin resistancegene and ColE1 origin permits selection and maintenance of the plasmidin E. coli. FIG. 19 shows expression of 158P3D2.pcDNA3.1/mychis intransiently transfected 293T cells.

pcDNA3.1/CT-GFP-TOPO Construct: To express 158P3D2 in mammalian cellsand to allow detection of the recombinant proteins using fluorescence, a158P3D2 ORF, or portions thereof, with a consensus Kozak translationinitiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA).Protein expression is driven from the cytomegalovirus (CMV) promoter.The recombinant proteins have the Green Fluorescent Protein (GFP) fusedto the carboxyl-terminus facilitating non-invasive, in vivo detectionand cell biology studies. The pcDNA3.1CT-GFP-TOPO vector also containsthe bovine growth hormone (BGH) polyadenylation signal and transcriptiontermination sequence to enhance mRNA stability along with the SV40origin for episomal replication and simple vector rescue in cell linesexpressing the large T antigen. The Neomycin resistance gene allows forselection of mammalian cells that express the protein, and theampicillin resistance gene and ColE1 origin permits selection andmaintenance of the plasmid in E. coli. Additional constructs with anamino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning theentire length of a 158P3D2 protein.

PAPtag: A 158P3D2 ORF, or portions thereof, is cloned into pAPtag-5(GenHunter Corp. Nashville, Tenn.). This construct generates an alkalinephosphatase fusion at the carboxyl-terminus of a 158P3D2 protein whilefusing the IgGK signal sequence to the amino-terminus. Constructs arealso generated in which alkaline phosphatase with an amino-terminal IgGKsignal sequence is fused to the amino-terminus of a 158P3D2 protein. Theresulting recombinant 158P3D2 proteins are optimized for secretion intothe media of transfected mammalian cells and can be used to identifyproteins such as ligands or receptors that interact with 158P3D2proteins. Protein expression is driven from the CMV promoter and therecombinant proteins also contain myc and 6×His epitopes fused at thecarboxyl-terminus that facilitates detection and purification. TheZeocin resistance gene present in the vector allows for selection ofmammalian cells expressing the recombinant protein and the ampicillinresistance gene permits selection of the plasmid in E. coli.

ptag5: A 158P3D2 ORF, or portions thereof, is cloned into pTag-5. Thisvector is similar to pAPtag but without the alkaline phosphatase fusion.This construct generates 158P3D2 protein with an amino-terminal IgGKsignal sequence and myc and 6×His epitope tags at the carboxyl-terminusthat facilitate detection and affinity purification. The resultingrecombinant 158P3D2 protein is optimized for secretion into the media oftransfected mammalian cells, and is used as immunogen or ligand toidentify proteins such as ligands or receptors that interact with the158P3D2 proteins. Protein expression is driven from the CMV promoter.The Zeocin resistance gene present in the vector allows for selection ofmammalian cells expressing the protein, and the ampicillin resistancegene permits selection of the plasmid in E. coli.

PsecFc: A 158P3D2 ORF, or portions thereof, is also cloned into psecFc.The psecFc vector was assembled by cloning the human immunoglobulin G1(IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen,California). This construct generates an IgG 1 Fc fusion at thecarboxyl-terminus of the 158P3D2 proteins, while fusing the IgGK signalsequence to N-terminus. 158P3D2 fusions utilizing the murine IgG1 Fcregion are also used. The resulting recombinant 158P3D2 proteins areoptimized for secretion into the media of transfected mammalian cells,and can be used as immunogens or to identify proteins such as ligands orreceptors that interact with 158P3D2 protein. Protein expression isdriven from the CMV promoter. The hygromycin resistance gene present inthe vector allows for selection of mammalian cells that express therecombinant protein, and the ampicillin resistance gene permitsselection of the plasmid in E. coli.

pSRα Constructs: To generate mammalian cell lines that express 158P3D2constitutively, 158P3D2 ORF, or portions thereof, of 158P3D2 were clonedinto pSRα constructs. Amphotropic and ecotropic retroviruses weregenerated by transfection of pSRα constructs into the 293T-10A 1packaging line or co-transfection of pSRα and a helper plasmid(containing deleted packaging sequences) into the 293 cells,respectively. The retrovirus is used to infect a variety of mammaliancell lines, resulting in the integration of the cloned gene, 158P3D2,into the host cell-lines. Protein expression is driven from a longterminal repeat (LTR). The Neomycin resistance gene present in thevector allows for selection of mammalian cells that express the protein,and the ampicillin resistance gene and ColE1 origin permit selection andmaintenance of the plasmid in E. coli. The retroviral vectors canthereafter be used for infection and generation of various cell linesusing, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.

Additional pSRα constructs are made that fuse an epitope tag such as theFLAG™ tag to the carboxyl-terminus of 158P3D2 sequences to allowdetection using anti-Flag antibodies. For example, the FLAG™ sequence 5′gat tac aag gat gac gac gat aag 3′ (SEQ ID NO: 103) is added to cloningprimer at the 3′ end of the ORF. Additional pSRα constructs are made toproduce both amino-terminal and carboxyl-terminal GFP and myc/6×Hisfusion proteins of the full-length 158P3D2 proteins.

Additional Viral Vectors: Additional constructs are made forviral-mediated delivery and expression of 158P3D2. High virus titerleading to high level expression of 158P3D2 is achieved in viraldelivery systems such as adenoviral vectors and herpes amplicon vectors.A 158P3D2 coding sequences or fragments thereof are amplified by PCR andsubcloned into the AdEasy shuttle vector (Stratagene). Recombination andvirus packaging are performed according to the manufacturer'sinstructions to generate adenoviral vectors. Alternatively, 158P3D2coding sequences or fragments thereof are cloned into the HSV-1 vector(Imgenex) to generate herpes viral vectors. The viral vectors arethereafter used for infection of various cell lines such as PC3, NIH3T3, 293 or rat-1 cells.

Regulated Expression Systems: To control expression of 158P3D2 inmammalian cells, coding sequences of 158P3D2, or portions thereof, arecloned into regulated mammalian expression systems such as the T-RexSystem (Invitrogen), the GeneSwitch System (Invitrogen) and thetightly-regulated Ecdysone System (Sratagene). These systems allow thestudy of the temporal and concentration dependent effects of recombinant158P3D2. These vectors are thereafter used to control expression of158P3D2 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus Expression Systems

To generate recombinant 158P3D2 proteins in a baculovirus expressionsystem, 158P3D2 ORF, or portions thereof, are cloned into thebaculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides aHis-tag at the N-terminus. Specifically, pBlueBac-158P3D2 isco-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9(Spodoptera frugiperda) insect cells to generate recombinant baculovirus(see Invitrogen instruction manual for details). Baculovirus is thencollected from cell supernatant and purified by plaque assay.

Recombinant 158P3D2 protein is then generated by infection of HighFiveinsect cells (Invitrogen) with purified baculovirus. Recombinant 158P3D2protein can be detected using anti-158P3D2 or anti-His-tag antibody.158P3D2 protein can be purified and used in various cell-based assays oras immunogen to generate polyclonal and monoclonal antibodies specificfor 158P3D2 which are used for diagnostic and therapeutic purposes.

Example 9 Antigenicity Profiles and Secondary Structure

FIG. 5A-I, FIG. 6A-I, FIG. 7A-I, FIG. 8A-I, and FIG. 9A-I depictgraphically five amino acid profiles of 158P3D2 variants 1, 2a, 2b, 5a,14, 15, 16, 17, 18, (A) through (I) respectively, each assessmentavailable by accessing the ProtScale website on the ExPasy molecularbiology server.

These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R., 1981.Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6, Hydropathicity,(Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7,Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG.8, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int.J. Pept. Protein Res. 32:242-255); FIG. 9, Beta-turn (Deleage, G., RouxB. 1987 Protein Engineering 1:289-294); and optionally others availablein the art, such as on the ProtScale website, were used to identifyantigenic regions of each of the 158P3D2 variant proteins. Each of theabove amino acid profiles of 158P3D2 variants were generated using thefollowing ProtScale parameters for analysis: 1) A window size of 9; 2)100% weight of the window edges compared to the window center; and, 3)amino acid profile values normalized to lie between 0 and 1.

Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and PercentageAccessible Residues (FIG. 7) profiles were used to determine stretchesof hydrophilic amino acids (i.e., values greater than 0.5 on theHydrophilicity and Percentage Accessible Residues profile, and valuesless than 0.5 on the Hydropathicity profile). Such regions are likely tobe exposed to the aqueous environment, be present on the surface of theprotein, and thus available for immune recognition, such as byantibodies.

Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profiles determinestretches of amino acids (i.e., values greater than 0.5 on the Beta-turnprofile and the Average Flexibility profile) that are not constrained insecondary structures such as beta sheets and alpha helices. Such regionsare also more likely to be exposed on the protein and thus accessible toimmune recognition, such as by antibodies.

Antigenic sequences of the 158P3D2 variant proteins indicated, e.g., bythe profiles set forth in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and/or FIG. 9are used to prepare immunogens, either peptides or nucleic acids thatencode them, to generate therapeutic and diagnostic anti-158P3D2antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or morethan 50 contiguous amino acids, or the corresponding nucleic acids thatencode them, from the 158P3D2 protein variants listed in FIGS. 2 and 3.In particular, peptide immunogens of the invention can comprise, apeptide region of at least 5 amino acids of FIGS. 2 and 3 in any wholenumber increment that includes an amino acid position having a valuegreater than 0.5 in the Hydrophilicity profiles of FIG. 5; a peptideregion of at least 5 amino acids of FIGS. 2 and 3 in any whole numberincrement that includes an amino acid position having a value less than0.5 in the Hydropathicity profile of FIG. 6; a peptide region of atleast 5 amino acids of FIGS. 2 and 3 in any whole number increment thatincludes an amino acid position having a value greater than 0.5 in thePercent Accessible Residues profiles of FIG. 7; a peptide region of atleast 5 amino acids of FIGS. 2 and 3 in any whole number increment thatincludes an amino acid position having a value greater than 0.5 in theAverage Flexibility profiles on FIG. 8; and, a peptide region of atleast 5 amino acids of FIGS. 2 and 3 in any whole number increment thatincludes an amino acid position having a value greater than 0.5 in theBeta-turn profile of FIG. 9. Peptide immunogens of the invention canalso comprise nucleic acids that encode any of the forgoing.

All immunogens of the invention, peptide or nucleic acid, can beembodied in human unit dose form, or comprised by a composition thatincludes a pharmaceutical excipient compatible with human physiology.

The secondary structures of 158P3D2 protein variants 1, 2a, 2b, 5a, 14,15, 16, 17, and 18, namely the predicted presence and location of alphahelices, extended strands, and random coils, are predicted from theirprimary amino acid sequences using the HNN—Hierarchical Neural Networkmethod (NPS@: Network Protein Sequence Analysis TIBS 2000 March Vol. 25,No 3 [291]:147-150 Combet C., Blanchet C., Geourjon C. and Deléage G.,accessed from the ExPasy molecular biology server. The analysisindicates that 158P3D2 variant 1 is composed of 32.93% alpha helix,18.29% extended strand, and 48.78% random coil (FIG. 13A). 158P3D2variant 2a is composed of 25.58% alpha helix, 18.22% extended strand,and 55.93% random coil (FIG. 13B). 158P3D2 variant 2b is composed of44.75% alpha helix, 11.60% extended strand, and 43.65% random coil (FIG.13C). 158P3D2 variant 5a is composed of 9.55% alpha helix, 26.40%extended strand, and 64.04% random coil (FIG. 13D). 158P3D2 variant 14is composed of 33.88% alpha helix, 13.42% extended strand, and 52.69%random coil (FIG. 13E). 158P3D2 variant 15 is composed of 33.28% alphahelix, 15.11% extended strand, and 51.62% random coil (FIG. 13F).158P3D2 variant 16 is composed of 32.76% alpha helix, 14.47% extendedstrand, and 52.76% random coil (FIG. 13G). 158P3D2 variant 17 iscomposed of 32.86% alpha helix, 14.69% extended strand, and 52.46%random coil (FIG. 13H). 158P3D2 variant 18 is composed of 27.21% alphahelix, 14.75% extended strand, and 58.03% random coil (FIG. 13I).

Analysis for the potential presence of transmembrane domains in the158P3D2 variant proteins 1, 2a, 2b, 5a, 14, 15, 16, 17, and 18, wascarried out using a variety of transmembrane prediction algorithmsaccessed from the ExPasy molecular biology server. Shown graphically inFIGS. 13L, 13N, 13P, 13R, 13T, 13V, 13X, 13Z are the results of analysisof variants 1, 2a, 2b, 5a, 14, 15, 16, 17, and 18, respectively, usingthe TMpred program. Shown graphically in FIGS. 13K, 13M, 13O, 13Q, 13S,13U, 13W, 13Y, 13AA are the results of analysis of variants 1, 2a, 2b,5a, 14, 15, 16, 17, and 18, respectively using the TMHMM program. Bothprograms predict the presence of 1 transmembrane domain in variant 1,Both programs predict that variants 2a, 2b, 5a, and 18 lacktransmembrane domains and are soluble proteins. The TMpred programpredicts that variants 14, 15, 16, and 17 have 2 transmembrane domainsof which the more carboxy-terminal transmembrane has a higherprobability of existence. The TMHMM program predicts that variants 14and 15 do not encode transmembrane domains and variants 16 and 17contain 1 transmembrane domain. Analyses of the variants using otherstructural prediction programs are summarized in Table VI and Table L.

Example 10 Generation of 158P3D2 Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Inaddition to immunizing with a full length 158P3D2 protein variant,computer algorithms are employed in design of immunogens that, based onamino acid sequence analysis contain characteristics of being antigenicand available for recognition by the immune system of the immunized host(see the Example entitled “Antigenicity Profiles and SecondaryStructure”). Such regions would be predicted to be hydrophilic,flexible, in beta-turn conformations, and be exposed on the surface ofthe protein (see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9 foramino acid profiles that indicate such regions of 158P3D2 proteinvariant 1 or other protein variants).

For example, recombinant bacterial fusion proteins or peptidescontaining hydrophilic, flexible, beta-turn regions of 158P3D2 proteinvariants are used as antigens to generate polyclonal antibodies in NewZealand White rabbits or monoclonal antibodies as described in Example11 (“Generation of Monoclonal Antibodies”). For example, in 158P3D2variant 1, such regions include, but are not limited to, amino acids1-25, amino acids 37-54, amino acids 60-73, amino acids 187-225, andamino acids 235-271. An extracellular epitope peptide encoding aminoacids 315 to 328 is also used to generate antibodies that bind to theextracellular region of 158P3D2 protein. It is useful to conjugate theimmunizing agent to a protein known to be immunogenic in the mammalbeing immunized. Examples of such immunogenic proteins include, but arenot limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. In one embodiment, apeptide encoding amino acids 315-328 of 158P3D2 variant 1 was conjugatedto KLH and used to immunize a rabbit. Alternatively the immunizing agentmay include all or portions of the 158P3D2 variant proteins, analogs orfusion proteins thereof. For example, the 158P3D2 variant 1 amino acidsequence can be fused using recombinant DNA techniques to any one of avariety of fusion protein partners that are well known in the art, suchas glutathione-S-transferase (GST) and HIS tagged fusion proteins.

In one embodiment, amino acids 155-290 of 158P3D2 variant 1 were fusedto His using recombinant techniques and the pET21b expression vector. Inanother embodiment, amino acids 260-328 were cloned into the pET21bexpression vector. The proteins are then expressed, purified, and usedto immunize rabbits. Such fusion proteins are purified from inducedbacteria using the appropriate affinity matrix.

Other recombinant bacterial fusion proteins that may be employed includemaltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulinconstant region (see the section entitled “Production of 158P3D2 inProkaryotic Systems” and Current Protocols In Molecular Biology, Volume2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P.S.,Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L. (1991)J. Exp. Med. 174, 561-566).

In addition to bacterial derived fusion proteins, mammalian expressedprotein antigens are also used. These antigens are expressed frommammalian expression vectors such as the Tag5 and Fc-fusion vectors (seethe section entitled “Production of Recombinant 158P3D2 in EukaryoticSystems”), and retain post-translational modifications such asglycosylations found in native protein. In one embodiment, amino acids1-236 of 158P3D2 variant 2a is cloned into the Tag5 mammalian secretionvector, and expressed in 293T cells. The recombinant protein is purifiedby metal chelate chromatography from tissue culture supernatants of 293Tcells stably expressing the recombinant vector. The purified Tag5158P3D2 protein is then used as immunogen.

During the immunization protocol, it is useful to mix or emulsify theantigen in adjuvants that enhance the immune response of the hostanimal. Examples of adjuvants include, but are not limited to, completeFreund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneouslywith up to 200 μg, typically 100-200 μg, of fusion protein or peptideconjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits arethen injected subcutaneously every two weeks with up to 200 μg,typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant(IFA). Test bleeds are taken approximately 7-10 days following eachimmunization and used to monitor the titer of the antiserum by ELISA.

To test reactivity and specificity of immune serum, such a rabbit serumderived from immunization with the His-fusion of 158P3D2 variant 1protein, the full-length 158P3D2 variant 1 cDNA was cloned into pcDNA3.1 myc-his expression vector (Invitrogen, see the Example entitled“Production of Recombinant 158P3D2 in Eukaryotic Systems”). Aftertransfection of the constructs into 293T cells, cell lysates are probedwith the anti-158P3D2 serum and with anti-His antibody (Santa CruzBiotechnologies, Santa Cruz, Calif.) to determine specific reactivity todenatured 158P3D2 protein using the Western blot technique. In addition,the immune serum is tested by fluorescence microscopy, flow cytometryand immunoprecipitation against 293T and other recombinant158P3D2-expressing cells to determine specific recognition of nativeprotein. Western blot, immunoprecipitation, fluorescent microscopy, andflow cytometric techniques using cells that endogenously express 158P3D2are also carried out to test reactivity and specificity.

Anti-serum from rabbits immunized with 158P3D2 variant fusion proteins,such as GST and MBP fusion proteins, are purified by depletion ofantibodies reactive to the fusion partner sequence by passage over anaffinity column containing the fusion partner either alone or in thecontext of an irrelevant fusion protein. For example, antiserum derivedfrom a GST-158P3D2 variant 1 fusion protein is first purified by passageover a column of GST protein covalently coupled to AffiGel matrix(BioRad, Hercules, Calif.). The antiserum is then affinity purified bypassage over a column composed of a MBP-158P3D2 fusion proteincovalently coupled to Affigel matrix. The serum is then further purifiedby protein G affinity chromatography to isolate the IgG fraction. Serafrom other His-tagged antigens and peptide immunized rabbits as well asfusion partner depleted sera are affinity purified by passage over acolumn matrix composed of the original protein immunogen or freepeptide.

Example 11 Generation of 158P3D2 Monoclonal Antibodies (mAbs)

In one embodiment, therapeutic mAbs to 158P3D2 variants comprise thosethat react with epitopes specific for each variant protein or specificto sequences in common between the variants that would disrupt ormodulate the biological function of the 158P3D2 variants, for examplethose that would disrupt the interaction with ligands and bindingpartners. Immunogens for generation of such mAbs include those designedto encode or contain the entire 158P3D2 protein variant sequence,regions predicted to contain functional motifs, and regions of the158P3D2 protein variants predicted to be antigenic from computeranalysis of the amino acid sequence (see, e.g., FIG. 5, FIG. 6, FIG. 7,FIG. 8, or FIG. 9, and the Example entitled “Antigenicity Profiles andSecondary Structure”). Immunogens include peptides, recombinantbacterial proteins, and mammalian expressed Tag 5 proteins and human andmurine IgG FC fusion proteins. In addition, cells engineered to expresshigh levels of a respective 158P3D2 variant, such as Rat1-158P3D2variant 1 or 300.19-158P3D2 variant 1 murine Pre-B cells, were used toimmunize mice.

To generate mAbs to a 158P3D2 variant, mice are first immunizedintraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or10⁷ 158P3D2-expressing cells mixed in complete Freund's adjuvant. Miceare then subsequently immunized IP every 2-4 weeks with, typically,10-50 μg of protein immunogen or 10⁷ cells mixed in incomplete Freund'sadjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. Inaddition to the above protein and cell-based immunization strategies, aDNA-based immunization protocol is employed in which a mammalianexpression vector encoding a 158P3D2 variant sequence is used toimmunize mice by direct injection of the plasmid DNA. For example, aminoacids 1-400 of 158P3D2 of variant 15 is cloned into the Tag5 mammaliansecretion vector and the recombinant vector will then be used asimmunogen. In another example the same amino acids are cloned into anFc-fusion secretion vector in which the 158P3D2 variant 15 sequence isfused at the amino-terminus to an IgK leader sequence and at thecarboxyl-terminus to the coding sequence of the human or murine IgG Fcregion. This recombinant vector is then used as immunogen. The plasmidimmunization protocols are used in combination with purified proteinsexpressed from the same vector and with cells expressing the respective158P3D2 variant.

During the immunization protocol, test bleeds are taken 7-10 daysfollowing an injection to monitor titer and specificity of the immuneresponse. Once appropriate reactivity and specificity is obtained asdetermined by ELISA, Western blotting, immunoprecipitation, fluorescencemicroscopy, and flow cytometric analyses, fusion and hybridomageneration is then carried out with established procedures well known inthe art (see, e.g., Harlow and Lane, 1988).

In one embodiment for generating 158P3D2 monoclonal antibodies, apeptide encoding amino acids 315-328 of 158P3D2 variant 1 was coupled toKLH and use to immunize mice. Balb C mice were immunized with 10 μg ofthe KLH-peptide mixed in adjuvant. Mice were subsequently immunized overseveral weeks with the KLH-peptide. ELISA using the peptide coupled to adifferent carrier, ovalbumin, determined the titer of serum from theimmunized mice (See FIG. 29). Reactivity and specificity of the serum tofull length 158P3D2 variant 1 protein was monitored by flow cytometryand Western blotting using recombinant 158P3D2 variant 1-expressingcells and cells endogenously expressing 158P3D2 variant 1 protein. Miceshowing the strongest reactivity were rested and given a final injectionof antigen and sacrificed for fusion. The lymph nodes of the sacrificedmice were harvested and fused to SPO/2 myeloma cells using standardprocedures (Harlow and Lane, 1988). Supernatants from HAT selectedgrowth wells were analyzed by flow cytometry to identifyspecific-158P3D2 surface binding MAbs. Supernatants were also screenedby ELISA, Western blot, immunoprecipitation, and fluorescent microscopyto identify 158P3D2 specific antibody-producing clones.

In other embodiments, 158P3D2 variant specific MAbs are generated byemploying immunogens that encode amino acid sequences unique to eachvariant or created by unique junctions from alternative splicing ofexons. For example, a peptide encoding amino acids 1018-1035 of 158P3D2variant 15 is coupled to KLH and used to immunize mice. In anotherexample, amino acids 1375-1393 of 158P3D2 variant 14 is coupled to KLHand used to immunize mice. Hybridomas resulting from fusion of theB-cells from the mice are screened on cells expressing the respective158P3D2 variant protein from which the antigen was derived andcross-screened on cells expressing the other variant proteins toidentify variant specific MAbs and MAbs that may recognize more than 1variant.

The binding affinity of 158P3D2 variant specific monoclonal antibodieswas determined using standard technologies. Affinity measurementsquantify the strength of antibody to epitope binding and are used tohelp define which 158P3D2 variant monoclonal antibodies preferred fordiagnostic or therapeutic use, as appreciated by one of skill in theart. The BIAcore system (Uppsala, Sweden) is a preferred method fordetermining binding affinity. The BIAcore system uses surface plasmonresonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton andMyszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecularinteractions in real time. BIAcore analysis conveniently generatesassociation rate constants, dissociation rate constants, equilibriumdissociation constants, and affinity constants.

In addition, equilibrium binding analysis of a dilution series of theMAb was also used to determine affinity defined by the dissociationconstant (KD). The KD is determined by non-linear regression of theequilibrium binding data of the concentration series. The KD is definedas the concentration at which half-maximal binding of the MAb to theantigen is attained under equilibrium conditions.

Example 12 HLA Class I and Class II Binding Assays

HLA class I and class II binding assays using purified HLA molecules areperformed in accordance with disclosed protocols (e.g., PCT publicationsWO 94/20127 and WO 94/03205; Sidney et al., Current Protocols inImmunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995);Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHCmolecules (5 to 500 nM) are incubated with various unlabeled peptideinhibitors and 1-10 nM 125I-radiolabeled probe peptides as described.Following incubation, MHC-peptide complexes are separated from freepeptide by gel filtration and the fraction of peptide bound isdetermined. Typically, in preliminary experiments, each MHC preparationis titered in the presence of fixed amounts of radiolabeled peptides todetermine the concentration of HLA molecules necessary to bind 10-20% ofthe total radioactivity. All subsequent inhibition and direct bindingassays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC50≧[HLA], the measuredIC50 values are reasonable approximations of the true KD values. Peptideinhibitors are typically tested at concentrations ranging from 120 μg/mlto 1.2 ng/ml, and are tested in two to four completely independentexperiments. To allow comparison of the data obtained in differentexperiments, a relative binding figure is calculated for each peptide bydividing the IC50 of a positive control for inhibition by the IC50 foreach tested peptide (typically unlabeled versions of the radiolabeledprobe peptide). For database purposes, and inter-experiment comparisons,relative binding values are compiled. These values can subsequently beconverted back into IC50 nM values by dividing the IC50 nM of thepositive controls for inhibition by the relative binding of the peptideof interest. This method of data compilation is accurate and consistentfor comparing peptides that have been tested on different days, or withdifferent lots of purified MHC.

Binding assays as outlined above may be used to analyze HLA supermotifand/or HLA motif-bearing peptides (see Table IV).

Example 13 Identification of HLA Supermotif- and Motif-Bearing CTLCandidate Epitopes

HLA vaccine compositions of the invention can include multiple epitopes.The multiple epitopes can comprise multiple HLA supermotifs or motifs toachieve broad population coverage. This example illustrates theidentification and confirmation of supermotif- and motif-bearingepitopes for the inclusion in such a vaccine composition. Calculation ofpopulation coverage is performed using the strategy described below.

Computer Searches and Algorithms for Identification of Supermotif and/orMotif-Bearing Epitopes

The searches performed to identify the motif-bearing peptide sequencesin the Example entitled “Antigenicity Profiles” and Tables VIII-XXI andXXII-XLIX employ the protein sequence data from the gene product of158P3D2 set forth in FIGS. 2 and 3, the specific search peptides used togenerate the tables are listed in Table VII.

Computer searches for epitopes bearing HLA Class I or Class IIsupermotifs or motifs are performed as follows. All translated 158P3D2protein sequences are analyzed using a text string search softwareprogram to identify potential peptide sequences containing appropriateHLA binding motifs; such programs are readily produced in accordancewith information in the art in view of known motif/supermotifdisclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored usingpolynomial algorithms to predict their capacity to bind to specificHLA-Class I or Class II molecules. These polynomial algorithms accountfor the impact of different amino acids at different positions, and areessentially based on the premise that the overall affinity (or AG) ofpeptide-HLA molecule interactions can be approximated as a linearpolynomial function of the type:“ΔG”=ali×a2i×a3i . . . . ×ani

-   -   where aji is a coefficient which represents the effect of the        presence of a given amino acid (j) at a given position (i) along        the sequence of a peptide of n amino acids. The crucial        assumption of this method is that the effects at each position        are essentially independent of each other (i.e., independent        binding of individual side-chains). When residue j occurs at        position i in the peptide, it is assumed to contribute a        constant amount ji to the free energy of binding of the peptide        irrespective of the sequence of the rest of the peptide.

The method of derivation of specific algorithm coefficients has beendescribed in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (seealso Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al.,J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchorand non-anchor alike, the geometric mean of the average relative binding(ARB) of all peptides carrying j is calculated relative to the remainderof the group, and used as the estimate of ji. For Class II peptides, ifmultiple alignments are possible, only the highest scoring alignment isutilized, following an iterative procedure. To calculate an algorithmscore of a given peptide in a test set, the ARB values corresponding tothe sequence of the peptide are multiplied. If this product exceeds achosen threshold, the peptide is predicted to bind. Appropriatethresholds are chosen as a function of the degree of stringency ofprediction desired.

Selection of HLA-A2 Supertype Cross-Reactive Peptides

Protein sequences from 158P3D2 are scanned utilizing motifidentification software, to identify 8-, 9-10- and 11-mer sequencescontaining the HLA-A2-supermotif main anchor specificity. Typically,these sequences are then scored using the protocol described above andthe peptides corresponding to the positive-scoring sequences aresynthesized and tested for their capacity to bind purified HLA-A*0201molecules in vitro (HLA-A*0201 is considered a prototype A2 supertypemolecule).

These peptides are then tested for the capacity to bind to additionalA2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptidesthat bind to at least three of the five A2-supertype alleles tested aretypically deemed A2-supertype cross-reactive binders. Preferred peptidesbind at an affinity equal to or less than 500 nM to three or more HLA-A2supertype molecules.

Selection of HLA-A3 Supermotif-Bearing Epitopes

The 158P3D2 protein sequence(s) scanned above is also examined for thepresence of peptides with the HLA-A3-supermotif primary anchors.Peptides corresponding to the HLA A3 supermotif-bearing sequences arethen synthesized and tested for binding to HLA-A*0301 and HLA-A*1101molecules, the molecules encoded by the two most prevalent A3-supertypealleles. The peptides that bind at least one of the two alleles withbinding affinities of ≦500 nM, often ≦200 nM, are then tested forbinding cross-reactivity to the other common A3-supertype alleles (e.g.,A*3101, A*3301, and A*6801) to identify those that can bind at leastthree of the five HLA-A3-supertype molecules tested.

Selection of HLA-B7 Supermotif Bearing Epitopes

The 158P3D2 protein(s) scanned above is also analyzed for the presenceof 8-, 9-10-, or 11-mer peptides with the HLA-B7-supermotif.Corresponding peptides are synthesized and tested for binding toHLA-B*0702, the molecule encoded by the most common B7-supertype allele(i.e., the prototype B7 supertype allele). Peptides binding B*0702 withIC50 of ≦500 nM are identified using standard methods. These peptidesare then tested for binding to other common B7-supertype molecules(e.g., B*3501, B*5101, B*5301, and B*5401). Peptides capable of bindingto three or more of the five B7-supertype alleles tested are therebyidentified.

Selection of A1 and A24 Motif-Bearing Epitopes

To further increase population coverage, HLA-A1 and -A24 epitopes canalso be incorporated into vaccine compositions. An analysis of the158P3D2 protein can also be performed to identify HLA-A1- andA24-motif-containing sequences.

High affinity and/or cross-reactive binding epitopes that bear othermotif and/or supermotifs are identified using analogous methodology.

Example 14 Confirmation of Immunogenicity

Cross-reactive candidate CTL A2-supermotif-bearing peptides that areidentified as described herein are selected to confirm in vitroimmunogenicity. Confirmation is performed using the followingmethodology:

Target Cell Lines for Cellular Screening:

The 0.221A2.1 cell line, produced by transferring the HLA-A2.1 gene intothe HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221,is used as the peptide-loaded target to measure activity ofHLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 mediumsupplemented with antibiotics, sodium pyruvate, nonessential amino acidsand 10% (v/v) heat inactivated FCS. Cells that express an antigen ofinterest, or transfectants comprising the gene encoding the antigen ofinterest, can be used as target cells to confirm the ability ofpeptide-specific CTLs to recognize endogenous antigen.

Primary CTL Induction Cultures:

Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640plus 5% AB human serum, non-essential amino acids, sodium pyruvate,L-glutamine and penicillin/streptomycin). The monocytes are purified byplating 10×106 PBMC/well in a 6-well plate. After 2 hours at 37° C., thenon-adherent cells are removed by gently shaking the plates andaspirating the supernatants. The wells are washed a total of three timeswith 3 ml RPMI to remove most of the non-adherent and loosely adherentcells. Three ml of complete medium containing 50 ng/ml of GM-CSF and1,000 U/ml of IL-4 are then added to each well. TNFα is added to the DCson day 6 at 75 ng/ml and the cells are used for CTL induction cultureson day 7.

Induction of CTL with DC and Peptide: CD8+ T-cells are isolated bypositive selection with Dynal immunomagnetic beads (Dynabeads® M-450)and the detacha-bead® reagent. Typically about 200-250×10⁶ PBMC areprocessed to obtain 24×10⁶ CD8+ T-cells (enough for a 48-well plateculture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse,washed once with PBS containing 1% human AB serum and resuspended inPBS/1% AB serum at a concentration of 20×106 cells/ml. The magneticbeads are washed 3 times with PBS/AB serum, added to the cells (140 μlbeads/20×106 cells) and incubated for 1 hour at 4° C. with continuousmixing. The beads and cells are washed 4× with PBS/AB serum to removethe nonadherent cells and resuspended at 100×106 cells/ml (based on theoriginal cell number) in PBS/AB serum containing 100 μl/ml detacha-bead®reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at roomtemperature with continuous mixing. The beads are washed again withPBS/AB/DNAse to collect the CD8+ T-cells. The DC are collected andcentrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1%BSA, counted and pulsed with μg/ml of peptide at a cell concentration of1-2×106/ml in the presence of 3 μg/ml B2-microglobulin for 4 hours at20° C. The DC are then irradiated (4,200 rads), washed 1 time withmedium and counted again.

Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1×105cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×10⁶cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml ofIL-7. Recombinant human IL-10 is added the next day at a finalconcentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10IU/ml.

Restimulation of the induction cultures with peptide-pulsed adherentcells: Seven and fourteen days after the primary induction, the cellsare restimulated with peptide-pulsed adherent cells. The PBMCs arethawed and washed twice with RPMI and DNAse. The cells are resuspendedat 5×106 cells/ml and irradiated at ˜4200 rads. The PBMCs are plated at2×106 in 0.5 ml complete medium per well and incubated for 2 hours at37° C. The plates are washed twice with RPMI by tapping the plate gentlyto remove the nonadherent cells and the adherent cells pulsed with 10μg/ml of peptide in the presence of 3 μg/ml β2 microglobulin in 0.25 mlRPMI/5% AB per well for 2 hours at 37° C. Peptide solution from eachwell is aspirated and the wells are washed once with RPMI. Most of themedia is aspirated from the induction cultures (CD8+ cells) and broughtto 0.5 ml with fresh media. The cells are then transferred to the wellscontaining the peptide-pulsed adherent cells. Twenty four hours laterrecombinant human IL-10 is added at a final concentration of 10 ng/mland recombinant human IL2 is added the next day and again 2-3 days laterat 50 IU/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75,1998). Seven days later, the cultures are assayed for CTL activity in a51 Cr release assay. In some experiments the cultures are assayed forpeptide-specific recognition in the in situ IFNγ ELISA at the time ofthe second restimulation followed by assay of endogenous recognition 7days later. After expansion, activity is measured in both assays for aside-by-side comparison.

Measurement of CTL Lytic Activity by 51Cr Release.

Seven days after the second restimulation, cytotoxicity is determined ina standard (5 hr) 51Cr release assay by assaying individual wells at asingle E:T. Peptide-pulsed targets are prepared by incubating the cellswith 10 μg/ml peptide overnight at 37° C.

Adherent target cells are removed from culture flasks with trypsin-EDTA.Target cells are labeled with 200 μCi of 51Cr sodium chromate (Dupont,Wilmington, Del.) for 1 hour at 37° C. Labeled target cells areresuspended at 106 per ml and diluted 1:10 with K562 cells at aconcentration of 3.3×106/ml (an NK-sensitive erythroblastoma cell lineused to reduce non-specific lysis). Target cells (100 μl) and effectors(10011) are plated in 96 well round-bottom plates and incubated for 5hours at 37° C. At that time, 100 μl of supernatant are collected fromeach well and percent lysis is determined according to the formula:[(cpm of the test sample−cpm of the spontaneous 51Cr releasesample)/(cpm of the maximal 51Cr release sample−cpm of the spontaneous51Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeledtargets with 1% Triton X-100 and media alone, respectively. A positiveculture is defined as one in which the specific lysis(sample-background) is 10% or higher in the case of individual wells andis 15% or more at the two highest E:T ratios when expanded cultures areassayed.

In Situ Measurement of Human IFNγ Production as an Indicator ofPeptide-Specific and Endogenous Recognition

Immulon 2 plates are coated with mouse anti-human IFNγ monoclonalantibody (4 μg/ml 0.1M NaHCO3, pH8.2) overnight at 4° C. The plates arewashed with Ca2+, Mg2+-free PBS/0.05% Tween 20 and blocked with PBS/10%FCS for two hours, after which the CTLs (100 μl/well) and targets (100l/well) are added to each well, leaving empty wells for the standardsand blanks (which received media only). The target cells, eitherpeptide-pulsed or endogenous targets, are used at a concentration of1×106 cells/ml. The plates are incubated for 48 hours at 37° C. with 5%CO₂.

Recombinant human IFN-gamma is added to the standard wells starting at400 pg or 1200 μg/100 microliter/well and the plate incubated for twohours at 37° C. The plates are washed and 100 μl of biotinylated mouseanti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2 hours at roomtemperature. After washing again, 100 microliter HRP-streptavidin(1:4000) are added and the plates incubated for one hour at roomtemperature. The plates are then washed 6× with wash buffer, 100microliter/well developing solution (TMB 1:1) are added, and the platesallowed to develop for 5-15 minutes. The reaction is stopped with 50microliter/well 1M H3PO4 and read at OD450. A culture is consideredpositive if it measured at least 50 pg of IFN-gamma/well abovebackground and is twice the background level of expression.

CTL Expansion.

Those cultures that demonstrate specific lytic activity againstpeptide-pulsed targets and/or tumor targets are expanded over a two weekperiod with anti-CD3. Briefly, 5×10⁴ CD8+ cells are added to a T25 flaskcontaining the following: 1×106 irradiated (4,200 rad) PBMC (autologousor allogeneic) per ml, 2×105 irradiated (8,000 rad) EBV-transformedcells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640containing 10% (v/v) human AB serum, non-essential amino acids, sodiumpyruvate, 25 μM 2-mercaptoethanol, L-glutamine andpenicillin/streptomycin. Recombinant human IL2 is added 24 hours laterat a final concentration of 2001U/ml and every three days thereafterwith fresh media at 501U/ml. The cells are split if the cellconcentration exceeds 1×106/ml and the cultures are assayed between days13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the 51Cr release assayor at 1×106/ml in the in situ IFNγ assay using the same targets asbefore the expansion.

Cultures are expanded in the absence of anti-CD3+ as follows. Thosecultures that demonstrate specific lytic activity against peptide andendogenous targets are selected and 5×104 CD8+ cells are added to a T25flask containing the following: 1×106 autologous PBMC per ml which havebeen peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. andirradiated (4,200 rad); 2×105 irradiated (8,000 rad) EBV-transformedcells per ml RPMI-1640 containing 10% (v/v) human AB serum,non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine andgentamicin.

Immunogenicity of A2 Supermotif-Bearing Peptides

A2-supermotif cross-reactive binding peptides are tested in the cellularassay for the ability to induce peptide-specific CTL in normalindividuals. In this analysis, a peptide is typically considered to bean epitope if it induces peptide-specific CTLs in at least individuals,and preferably, also recognizes the endogenously expressed peptide.

Immunogenicity can also be confirmed using PBMCs isolated from patientsbearing a tumor that expresses 158P3D2. Briefly, PBMCs are isolated frompatients, re-stimulated with peptide-pulsed monocytes and assayed forthe ability to recognize peptide-pulsed target cells as well astransfected cells endogenously expressing the antigen.

Evaluation of A*03/A11 Immunogenicity

HLA-A3 supermotif-bearing cross-reactive binding peptides are alsoevaluated for immunogenicity using methodology analogous for that usedto evaluate the immunogenicity of the HLA-A2 supermotif peptides.

Evaluation of B7 Immunogenicity

Immunogenicity screening of the B7-supertype cross-reactive bindingpeptides identified as set forth herein are confirmed in a manneranalogous to the confirmation of A2- and A3-supermotif-bearing peptides.

Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24 etc.are also confirmed using similar methodology.

Example 15 Implementation of the Extended Supermotif to Improve theBinding Capacity of Native Epitopes by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondaryresidues) are useful in the identification and preparation of highlycross-reactive native peptides, as demonstrated herein. Moreover, thedefinition of HLA motifs and supermotifs also allows one to engineerhighly cross-reactive epitopes by identifying residues within a nativepeptide sequence which can be analoged to confer upon the peptidecertain characteristics, e.g. greater cross-reactivity within the groupof HLA molecules that comprise a supertype, and/or greater bindingaffinity for some or all of those HLA molecules. Examples of analogingpeptides to exhibit modulated binding affinity are set forth in thisexample.

Analoging at Primary Anchor Residues

Peptide engineering strategies are implemented to further increase thecross-reactivity of the epitopes. For example, the main anchors ofA2-supermotif-bearing peptides are altered, for example, to introduce apreferred L, I, V, or M at position 2, and I or V at the C-terminus.

To analyze the cross-reactivity of the analog peptides, each engineeredanalog is initially tested for binding to the prototype A2 supertypeallele A*0201, then, if A*0201 binding capacity is maintained, forA2-supertype cross-reactivity.

Alternatively, a peptide is confirmed as binding one or all supertypemembers and then analoged to modulate binding affinity to any one (ormore) of the supertype members to add population coverage.

The selection of analogs for immunogenicity in a cellular screeninganalysis is typically further restricted by the capacity of the parentwild type (WT) peptide to bind at least weakly, i.e., bind at an IC50 of500 nM or less, to three of more A2 supertype alleles. The rationale forthis requirement is that the WT peptides must be present endogenously insufficient quantity to be biologically relevant. Analoged peptides havebeen shown to have increased immunogenicity and cross-reactivity by Tcells specific for the parent epitope (see, e.g., Parkhurst et al., J.Immunol. 157:2539, 1996; and Pogue et al., Proc. Natl. Acad. Sci. USA92:8166, 1995).

In the cellular screening of these peptide analogs, it is important toconfirm that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, target cells that endogenouslyexpress the epitope.

Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides

Analogs of HLA-A3 supermotif-bearing epitopes are generated usingstrategies similar to those employed in analoging HLA-A2supermotif-bearing peptides. For example, peptides binding to 3/5 of theA3-supertype molecules are engineered at primary anchor residues topossess a preferred residue (V, S, M, or A) at position 2.

The analog peptides are then tested for the ability to bind A*03 andA*11 (prototype A3 supertype alleles). Those peptides that demonstrate≦500 nM binding capacity are then confirmed as having A3-supertypecross-reactivity.

Similarly to the A2- and A3-motif bearing peptides, peptides binding 3or more B7-supertype alleles can be improved, where possible, to achieveincreased cross-reactive binding or greater binding affinity or bindinghalf life. B7 supermotif-bearing peptides are, for example, engineeredto possess a preferred residue (V, I, L, or F) at the C-terminal primaryanchor position, as demonstrated by Sidney et al. (J. Immunol.157:3480-3490, 1996).

Analoging at primary anchor residues of other motif and/orsupermotif-bearing epitopes is performed in a like manner.

The analog peptides are then be confirmed for immunogenicity, typicallyin a cellular screening assay. Again, it is generally important todemonstrate that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, targets that endogenously expressthe epitope.

Analoging at Secondary Anchor Residues

Moreover, HLA supermotifs are of value in engineering highlycross-reactive peptides and/or peptides that bind HLA molecules withincreased affinity by identifying particular residues at secondaryanchor positions that are associated with such properties. For example,the binding capacity of a B7 supermotif-bearing peptide with an Fresidue at position 1 is analyzed. The peptide is then analoged to, forexample, substitute L for F at position 1. The analoged peptide isevaluated for increased binding affinity, binding half life and/orincreased cross-reactivity. Such a procedure identifies analogedpeptides with enhanced properties.

Engineered analogs with sufficiently improved binding capacity orcross-reactivity can also be tested for immunogenicity inHLA-B7-transgenic mice, following for example, IFA immunization orlipopeptide immunization. Analoged peptides are additionally tested forthe ability to stimulate a recall response using PBMC from patients with158P3D2-expressing tumors.

Other Analoging Strategies

Another form of peptide analoging, unrelated to anchor positions,involves the substitution of a cysteine with α-amino butyric acid. Dueto its chemical nature, cysteine has the propensity to form disulfidebridges and sufficiently alter the peptide structurally so as to reducebinding capacity. Substitution of α-amino butyric acid for cysteine notonly alleviates this problem, but has been shown to improve binding andcrossbinding capabilities in some instances (see, e.g., the review bySette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I.Chen, John Wiley & Sons, England, 1999).

Thus, by the use of single amino acid substitutions, the bindingproperties and/or cross-reactivity of peptide ligands for HLA supertypemolecules can be modulated.

Example 16 Identification and Confirmation of 158P3D2-Derived Sequenceswith HLA-DR Binding Motifs

Peptide epitopes bearing an HLA class II supermotif or motif areidentified and confirmed as outlined below using methodology similar tothat described for HLA Class I peptides.

Selection of HLA-DR-Supermotif-Bearing Epitopes.

To identify 158P3D2-derived, HLA class II HTL epitopes, a 158P3D2antigen is analyzed for the presence of sequences bearing anHLA-DR-motif or supermotif. Specifically, 15-mer sequences are selectedcomprising a DR-supermotif, comprising a 9-mer core, and three-residueN- and C-terminal flanking regions (15 amino acids total).

Protocols for predicting peptide binding to DR molecules have beendeveloped (Southwood et al., J. Immunol. 160:3363-3373, 1998). Theseprotocols, specific for individual DR molecules, allow the scoring, andranking, of 9-mer core regions. Each protocol not only scores peptidesequences for the presence of DR-supermotif primary anchors (i.e., atposition 1 and position 6) within a 9-mer core, but additionallyevaluates sequences for the presence of secondary anchors. Usingallele-specific selection tables (see, e.g., Southwood et al., ibid.),it has been found that these protocols efficiently select peptidesequences with a high probability of binding a particular DR molecule.Additionally, it has been found that performing these protocols intandem, specifically those for DR1, DR4w4, and DR7, can efficientlyselect DR cross-reactive peptides.

The 158P3D2-derived peptides identified above are tested for theirbinding capacity for various common HLA-DR molecules. All peptides areinitially tested for binding to the DR molecules in the primary panel:DR1, DR4w4, and DR7. Peptides binding at least two of these three DRmolecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19, andDR9 molecules in secondary assays. Finally, peptides binding at leasttwo of the four secondary panel DR molecules, and thus cumulatively atleast four of seven different DR molecules, are screened for binding toDR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides bindingat least seven of the ten DR molecules comprising the primary,secondary, and tertiary screening assays are considered cross-reactiveDR binders. 158P3D2-derived peptides found to bind common HLA-DR allelesare of particular interest.

Selection of DR3 Motif Peptides

Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, andHispanic populations, DR3 binding capacity is a relevant criterion inthe selection of HTL epitopes. Thus, peptides shown to be candidates mayalso be assayed for their DR3 binding capacity. However, in view of thebinding specificity of the DR3 motif, peptides binding only to DR3 canalso be considered as candidates for inclusion in a vaccine formulation.

To efficiently identify peptides that bind DR3, target 158P3D2 antigensare analyzed for sequences carrying one of the two DR3-specific bindingmotifs reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). Thecorresponding peptides are then synthesized and confirmed as having theability to bind DR3 with an affinity of 1 μM or better, i.e., less than1 μM. Peptides are found that meet this binding criterion and qualify asHLA class II high affinity binders.

DR3 binding epitopes identified in this manner are included in vaccinecompositions with DR supermotif-bearing peptide epitopes.

Similarly to the case of HLA class I motif-bearing peptides, the classII motif-bearing peptides are analoged to improve affinity orcross-reactivity. For example, aspartic acid at position 4 of the 9-mercore sequence is an optimal residue for DR3 binding, and substitutionfor that residue often improves DR 3 binding.

Example 17 Immunogenicity of 158P3D2-Derived HTL Epitopes

This example determines immunogenic DR supermotif- and DR3 motif-bearingepitopes among those identified using the methodology set forth herein.

Immunogenicity of HTL epitopes are confirmed in a manner analogous tothe determination of immunogenicity of CTL epitopes, by assessing theability to stimulate HTL responses and/or by using appropriatetransgenic mouse models. Immunogenicity is determined by screening for:1.) in vitro primary induction using normal PBMC or 2.) recall responsesfrom patients who have 158P3D2-expressing tumors.

Example 18 Calculation of Phenotypic Frequencies of HLA-Supertypes inVarious Ethnic Backgrounds to Determine Breadth of Population Coverage

This example illustrates the assessment of the breadth of populationcoverage of a vaccine composition comprised of multiple epitopescomprising multiple supermotifs and/or motifs.

In order to analyze population coverage, gene frequencies of HLA allelesare determined. Gene frequencies for each HLA allele are calculated fromantigen or allele frequencies utilizing the binomial distributionformulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., Human Immunol.45:79-93, 1996). To obtain overall phenotypic frequencies, cumulativegene frequencies are calculated, and the cumulative antigen frequenciesderived by the use of the inverse formula [af=1−(1−Cgf)2].

Where frequency data is not available at the level of DNA typing,correspondence to the serologically defined antigen frequencies isassumed. To obtain total potential supertype population coverage nolinkage disequilibrium is assumed, and only alleles confirmed to belongto each of the supertypes are included (minimal estimates). Estimates oftotal potential coverage achieved by inter-loci combinations are made byadding to the A coverage the proportion of the non-A covered populationthat could be expected to be covered by the B alleles considered (e.g.,total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3,A11, A31, A*3301, and A*6801. Although the A3-like supertype may alsoinclude A34, A66, and A*7401, these alleles were not included in overallfrequency calculations. Likewise, confirmed members of the A2-likesupertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206,A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmedalleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601,B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, andB*5602).

Population coverage achieved by combining the A2-, A3- and B7-supertypesis approximately 86% in five major ethnic groups. Coverage may beextended by including peptides bearing the A1 and A24 motifs. Onaverage, A1 is present in 12% and A24 in 29% of the population acrossfive different major ethnic groups (Caucasian, North American Black,Chinese, Japanese, and Hispanic). Together, these alleles arerepresented with an average frequency of 39% in these same ethnicpopulations. The total coverage across the major ethnicities when A1 andA24 are combined with the coverage of the A2-, A3- and B7-supertypealleles is >95%, see, e.g., Table IV (G). An analogous approach can beused to estimate population coverage achieved with combinations of classII motif-bearing epitopes.

Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin. Invest.100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al.,J. Immunol. 159:1648, 1997) have shown that highly cross-reactivebinding peptides are almost always recognized as epitopes. The use ofhighly cross-reactive binding peptides is an important selectioncriterion in identifying candidate epitopes for inclusion in a vaccinethat is immunogenic in a diverse population.

With a sufficient number of epitopes (as disclosed herein and from theart), an average population coverage is predicted to be greater than 95%in each of five major ethnic populations. The game theory Monte Carlosimulation analysis, which is known in the art (see e.g., Osborne, M. J.and Rubinstein, A. “A course in game theory” MIT Press, 1994), can beused to estimate what percentage of the individuals in a populationcomprised of the Caucasian, North American Black, Japanese, Chinese, andHispanic ethnic groups would recognize the vaccine epitopes describedherein. A preferred percentage is 90%. A more preferred percentage is95%.

Example 19 CTL Recognition Of Endogenously Processed Antigens afterPriming

This example confirms that CTL induced by native or analoged peptideepitopes identified and selected as described herein recognizeendogenously synthesized, i.e., native antigens.

Effector cells isolated from transgenic mice that are immunized withpeptide epitopes, for example HLA-A2 supermotif-bearing epitopes, arere-stimulated in vitro using peptide-coated stimulator cells. Six dayslater, effector cells are assayed for cytotoxicity and the cell linesthat contain peptide-specific cytotoxic activity are furtherre-stimulated. An additional six days later, these cell lines are testedfor cytotoxic activity on ⁵¹Cr labeled Jurkat-A2.1/K^(b) target cells inthe absence or presence of peptide, and also tested on ⁵¹Cr labeledtarget cells bearing the endogenously synthesized antigen, i.e. cellsthat are stably transfected with 158P3D2 expression vectors.

The results demonstrate that CTL lines obtained from animals primed withpeptide epitope recognize endogenously synthesized 158P3D2 antigen. Thechoice of transgenic mouse model to be used for such an analysis dependsupon the epitope(s) that are being evaluated. In addition toHLA-A*0201/K^(b) transgenic mice, several other transgenic mouse modelsincluding mice with human A11, which may also be used to evaluate A3epitopes, and B7 alleles have been characterized and others (e.g.,transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 andHLA-DR3 mouse models have also been developed, which may be used toevaluate HTL epitopes.

Example 20 Activity Of CTL-HTL Conjugated Epitopes In Transgenic Mice

This example illustrates the induction of CTLs and HTLs in transgenicmice, by use of a 158P3D2-derived CTL and HTL peptide vaccinecompositions. The vaccine composition used herein comprise peptides tobe administered to a patient with a 158P3D2-expressing tumor. Thepeptide composition can comprise multiple CTL and/or HTL epitopes. Theepitopes are identified using methodology as described herein. Thisexample also illustrates that enhanced immunogenicity can be achieved byinclusion of one or more HTL epitopes in a CTL vaccine composition; sucha peptide composition can comprise an HTL epitope conjugated to a CTLepitope. The CTL epitope can be one that binds to multiple HLA familymembers at an affinity of 500 nM or less, or analogs of that epitope.The peptides may be lipidated, if desired.

Immunization procedures: Immunization of transgenic mice is performed asdescribed (Alexander et al., J. Immunol. 159:4753-4761, 1997). Forexample, A2/K^(b) mice, which are transgenic for the human HLA A2.1allele and are used to confirm the immunogenicity of HLA-A*0201 motif-or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously(base of the tail) with a 0.1 ml of peptide in Incomplete Freund'sAdjuvant, or if the peptide composition is a lipidated CTL/HTLconjugate, in DMSO/saline, or if the peptide composition is apolypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days afterpriming, splenocytes obtained from these animals are restimulated withsyngenic irradiated LPS-activated lymphoblasts coated with peptide.

Cell lines: Target cells for peptide-specific cytotoxicity assays areJurkat cells transfected with the HLA-A2.1/K^(b) chimeric gene (e.g.,Vitiello et al., J. Exp. Med. 173:1007, 1991).

In vitro CTL activation: One week after priming, spleen cells (30×10⁶cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000rads), peptide coated lymphoblasts (10×10⁶ cells/flask) in 10 ml ofculture medium/T25 flask. After six days, effector cells are harvestedand assayed for cytotoxic activity.

Assay for cytotoxic activity: Target cells (1.0 to 1.5×10⁶) areincubated at 37° C. in the presence of 200 μl of ⁵¹Cr. After 60 minutes,cells are washed three times and resuspended in R10 medium. Peptide isadded where required at a concentration of 1 μg/ml. For the assay, 10⁴⁵¹Cr-labeled target cells are added to different concentrations ofeffector cells (final volume of 200 μl) in U-bottom 96-well plates.After a six hour incubation period at 37° C., a 0.1 ml aliquot ofsupernatant is removed from each well and radioactivity is determined ina Micromedic automatic gamma counter. The percent specific lysis isdetermined by the formula: percent specific release=100×(experimentalrelease−spontaneous release)/(maximum release−spontaneous release). Tofacilitate comparison between separate CTL assays run under the sameconditions, % ⁵¹Cr release data is expressed as lytic units/10⁶ cells.One lytic unit is arbitrarily defined as the number of effector cellsrequired to achieve 30% lysis of 10,000 target cells in a six hour ⁵¹Crrelease assay. To obtain specific lytic units/10⁶, the lytic units/10⁶obtained in the absence of peptide is subtracted from the lyticunits/10⁶ obtained in the presence of peptide. For example, if 30% ⁵¹ Crrelease is obtained at the effector (E): target (T) ratio of 50:1 (i.e.,5×10⁵ effector cells for 10,000 targets) in the absence of peptide and5:1 (i.e., 5×10⁴ effector cells for 10,000 targets) in the presence ofpeptide, the specific lytic units would be: [( 1/50,000)−(1/500,000)]×10⁶=18 LU.

The results are analyzed to assess the magnitude of the CTL responses ofanimals injected with the immunogenic CTL/HTL conjugate vaccinepreparation and are compared to the magnitude of the CTL responseachieved using, for example, CTL epitopes as outlined above in theExample entitled “Confirmation of Immunogenicity.” Analyses similar tothis may be performed to conform the immunogenicity of peptideconjugates containing multiple CTL epitopes and/or multiple HTLepitopes. In accordance with these procedures, it is found that a CTLresponse is induced, and concomitantly that an HTL response is inducedupon administration of such compositions.

Example 21 Selection of CTL and HTL Epitopes for Inclusion in a158P3D2-Specific Vaccine

This example illustrates a procedure for selecting peptide epitopes forvaccine compositions of the invention. The peptides in the compositioncan be in the form of a nucleic acid sequence, either single or one ormore sequences (i.e., minigene) that encodes peptide(s), or can besingle and/or polyepitopic peptides.

The following principles are utilized when selecting a plurality ofepitopes for inclusion in a vaccine composition. Each of the followingprinciples is balanced in order to make the selection.

Epitopes are selected which, upon administration, mimic immune responsesthat are correlated with 158P3D2 clearance. The number of epitopes useddepends on observations of patients who spontaneously clear 158P3D2. Forexample, if it has been observed that patients who spontaneously clear158P3D2-expressing cells generate an immune response to at least three(3) epitopes from 158P3D2 antigen, then at least three epitopes shouldbe included for HLA class I. A similar rationale is used to determineHLA class II epitopes.

Epitopes are often selected that have a binding affinity of an IC₅₀ of500 nM or less for an HLA class I molecule, or for class II, an IC₅₀ of1000 nM or less; or HLA Class I peptides with high binding scores fromthe BIMAS web site, at URL bimas.dcrt.nih.gov/.

In order to achieve broad coverage of the vaccine through out a diversepopulation, sufficient supermotif bearing peptides, or a sufficientarray of allele-specific motif bearing peptides, are selected to givebroad population coverage. In one embodiment, epitopes are selected toprovide at least 80% population coverage. A Monte Carlo analysis, astatistical evaluation known in the art, can be employed to assessbreadth, or redundancy, of population coverage.

When creating polyepitopic compositions, or a minigene that encodessame, it is typically desirable to generate the smallest peptidepossible that encompasses the epitopes of interest. The principlesemployed are similar, if not the same, as those employed when selectinga peptide comprising nested epitopes. For example, a protein sequencefor the vaccine composition is selected because it has maximal number ofepitopes contained within the sequence, i.e., it has a highconcentration of epitopes. Epitopes may be nested or overlapping (i.e.,frame shifted relative to one another). For example, with overlappingepitopes, two 9-mer epitopes and one 10-mer epitope can be present in a10 amino acid peptide. Each epitope can be exposed and bound by an HLAmolecule upon administration of such a peptide. A multi-epitopic,peptide can be generated synthetically, recombinantly, or via cleavagefrom the native source. Alternatively, an analog can be made of thisnative sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes. This embodimentprovides for the possibility that an as yet undiscovered aspect ofimmune system processing will apply to the native nested sequence andthereby facilitate the production of therapeutic or prophylactic immuneresponse-inducing vaccine compositions. Additionally such an embodimentprovides for the possibility of motif-bearing epitopes for an HLA makeupthat is presently unknown. Furthermore, this embodiment (absent thecreating of any analogs) directs the immune response to multiple peptidesequences that are actually present in 158P3D2, thus avoiding the needto evaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing nucleic acid vaccine compositions.Related to this embodiment, computer programs can be derived inaccordance with principles in the art, which identify in a targetsequence, the greatest number of epitopes per sequence length.

A vaccine composition comprised of selected peptides, when administered,is safe, efficacious, and elicits an immune response similar inmagnitude to an immune response that controls or clears cells that bearor overexpress 158P3D2.

Example 22 Construction of “Minigene” Multi-Epitope DNA Plasmids

This example discusses the construction of a minigene expressionplasmid.

Minigene plasmids may, of course, contain various configurations of Bcell, CTL and/or HTL epitopes or epitope analogs as described herein.

A minigene expression plasmid typically includes multiple CTL and HTLpeptide epitopes. In the present example, HLA-A2, -A3, -B7supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearingpeptide epitopes are used in conjunction with DR supermotif-bearingepitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearingpeptide epitopes derived 158P3D2, are selected such that multiplesupermotifs/motifs are represented to ensure broad population coverage.Similarly, HLA class II epitopes are selected from 158P3D2 to providebroad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearingepitopes and HLA DR-3 motif-bearing epitopes are selected for inclusionin the minigene construct. The selected CTL and HTL epitopes are thenincorporated into a minigene for expression in an expression vector.

Such a construct may additionally include sequences that direct the HTLepitopes to the endoplasmic reticulum. For example, the Ii protein maybe fused to one or more HTL epitopes as described in the art, whereinthe CLIP sequence of the Ii protein is removed and replaced with an HLAclass II epitope sequence so that HLA class II epitope is directed tothe endoplasmic reticulum, where the epitope binds to an HLA class IImolecules.

This example illustrates the methods to be used for construction of aminigene-bearing expression plasmid. Other expression vectors that maybe used for minigene compositions are available and known to those ofskill in the art.

The minigene DNA plasmid of this example contains a consensus Kozaksequence and a consensus murine kappa Ig-light chain signal sequencefollowed by CTL and/or HTL epitopes selected in accordance withprinciples disclosed herein. The sequence encodes an open reading framefused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1Myc-His vector.

Overlapping oligonucleotides that can, for example, average about 70nucleotides in length with 15 nucleotide overlaps, are synthesized andHPLC-purified. The oligonucleotides encode the selected peptide epitopesas well as appropriate linker nucleotides, Kozak sequence, and signalsequence. The final multiepitope minigene is assembled by extending theoverlapping oligonucleotides in three sets of reactions using PCR. APerkin/Elmer 9600 PCR machine is used and a total of 30 cycles areperformed using the following conditions: 95° C. for 15 sec, annealingtemperature (5° below the lowest calculated Tm of each primer pair) for30 sec, and 72° C. for 1 min.

For example, a minigene is prepared as follows. For a first PCRreaction, 5 μg of each of two oligonucleotides are annealed andextended: In an example using eight oligonucleotides, i.e., four pairsof primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM(NH4)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% Triton X-100,100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. Thefull-length dimer products are gel-purified, and two reactionscontaining the product of 1+2 and 3+4, and the product of 5+6 and 7+8are mixed, annealed, and extended for 10 cycles. Half of the tworeactions are then mixed, and 5 cycles of annealing and extensioncarried out before flanking primers are added to amplify the full lengthproduct. The full-length product is gel-purified and cloned intopCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 23 The Plasmid Construct and the Degree to Which it InducesImmunogenicity

The degree to which a plasmid construct, for example a plasmidconstructed in accordance with the previous Example, is able to induceimmunogenicity is confirmed in vitro by determining epitope presentationby APC following transduction or transfection of the APC with anepitope-expressing nucleic acid construct. Such a study determines“antigenicity” and allows the use of human APC. The assay determines theability of the epitope to be presented by the APC in a context that isrecognized by a T cell by quantifying the density of epitope-HLA class Icomplexes on the cell surface. Quantitation can be performed by directlymeasuring the amount of peptide eluted from the APC (see, e.g., Sijts etal., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684,1989); or the number of peptide-HLA class I complexes can be estimatedby measuring the amount of lysis or lymphokine release induced bydiseased or transfected target cells, and then determining theconcentration of peptide necessary to obtain equivalent levels of lysisor lymphokine release (see, e.g., Kageyama et al., J. Immunol.154:567-576, 1995).

Alternatively, immunogenicity is confirmed through in vivo injectionsinto mice and subsequent in vitro assessment of CTL and HTL activity,which are analyzed using cytotoxicity and proliferation assays,respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761,1994.

For example, to confirm the capacity of a DNA minigene constructcontaining at least one HLA-A2 supermotif peptide to induce CTLs invivo, HLA-A2. ₁/Kb transgenic mice, for example, are immunizedintramuscularly with 100 μg of naked cDNA. As a means of comparing thelevel of CTLs induced by cDNA immunization, a control group of animalsis also immunized with an actual peptide composition that comprisesmultiple epitopes synthesized as a single polypeptide as they would beencoded by the minigene.

Splenocytes from immunized animals are stimulated twice with each of therespective compositions (peptide epitopes encoded in the minigene or thepolyepitopic peptide), then assayed for peptide-specific cytotoxicactivity in a ⁵¹Cr release assay. The results indicate the magnitude ofthe CTL response directed against the A2-restricted epitope, thusindicating the in vivo immunogenicity of the minigene vaccine andpolyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responsesdirected toward the HLA-A2 supermotif peptide epitopes as does thepolyepitopic peptide vaccine. A similar analysis is also performed usingother HLA-A3 and HLA-B7 transgenic mouse models to assess CTL inductionby HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is alsofound that the minigene elicits appropriate immune responses directedtoward the provided epitopes.

To confirm the capacity of a class II epitope-encoding minigene toinduce HTLs in vivo, DR transgenic mice, or for those epitopes thatcross react with the appropriate mouse MHC molecule, I-A^(b)-restrictedmice, for example, are immunized intramuscularly with 100 μg of plasmidDNA. As a means of comparing the level of HTLs induced by DNAimmunization, a group of control animals is also immunized with anactual peptide composition emulsified in complete Freund's adjuvant.CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunizedanimals and stimulated with each of the respective compositions(peptides encoded in the minigene). The HTL response is measured using a³H-thymidine incorporation proliferation assay, (see, e.g., Alexander etal. Immunity 1:751-761, 1994). The results indicate the magnitude of theHTL response, thus demonstrating the in vivo immunogenicity of theminigene.

DNA minigenes, constructed as described in the previous Example, canalso be confirmed as a vaccine in combination with a boosting agentusing a prime boost protocol. The boosting agent can consist ofrecombinant protein (e.g., Barnett et al., Aids Res. and HumanRetroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia,for example, expressing a minigene or DNA encoding the complete proteinof interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegahet al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael,Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature Med.5:526-34, 1999).

For example, the efficacy of the DNA minigene used in a prime boostprotocol is initially evaluated in transgenic mice. In this example,A2.1/K^(b) transgenic mice are immunized IM with 100 μg of a DNAminigene encoding the immunogenic peptides including at least one HLA-A2supermotif-bearing peptide. After an incubation period (ranging from 3-9weeks), the mice are boosted IP with 10⁷ pfu/mouse of a recombinantvaccinia virus expressing the same sequence encoded by the DNA minigene.Control mice are immunized with 100 μg of DNA or recombinant vacciniawithout the minigene sequence, or with DNA encoding the minigene, butwithout the vaccinia boost. After an additional incubation period of twoweeks, splenocytes from the mice are immediately assayed forpeptide-specific activity in an ELISPOT assay. Additionally, splenocytesare stimulated in vitro with the A2-restricted peptide epitopes encodedin the minigene and recombinant vaccinia, then assayed forpeptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It is found that the minigene utilized in a prime-boost protocol elicitsgreater immune responses toward the HLA-A2 supermotif peptides than withDNA alone. Such an analysis can also be performed using HLA-A11 orHLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 orHLA-B7 motif or supermotif epitopes. The use of prime boost protocols inhumans is described below in the Example entitled “Induction of CTLResponses Using a Prime Boost Protocol.”

Example 24 Peptide Compositions for Prophylactic Uses

Vaccine compositions of the present invention can be used to prevent158P3D2 expression in persons who are at risk for tumors that bear thisantigen. For example, a polyepitopic peptide epitope composition (or anucleic acid comprising the same) containing multiple CTL and HTLepitopes such as those selected in the above Examples, which are alsoselected to target greater than 80% of the population, is administeredto individuals at risk for a 158P3D2-associated tumor.

For example, a peptide-based composition is provided as a singlepolypeptide that encompasses multiple epitopes. The vaccine is typicallyadministered in a physiological solution that comprises an adjuvant,such as Incomplete Freunds Adjuvant. The dose of peptide for the initialimmunization is from about 1 to about 50,000 μg, generally 100-5,000 μg,for a 70 kg patient. The initial administration of vaccine is followedby booster dosages at 4 weeks followed by evaluation of the magnitude ofthe immune response in the patient, by techniques that determine thepresence of epitope-specific CTL populations in a PBMC sample.Additional booster doses are administered as required. The compositionis found to be both safe and efficacious as a prophylaxis against158P3D2-associated disease.

Alternatively, a composition typically comprising transfecting agents isused for the administration of a nucleic acid-based vaccine inaccordance with methodologies known in the art and disclosed herein.

Example 25 Polyepitopic Vaccine Compositions Derived from Native 158P3D2Sequences

A native 158P3D2 polyprotein sequence is analyzed, preferably usingcomputer algorithms defined for each class I and/or class II supermotifor motif, to identify “relatively short” regions of the polyprotein thatcomprise multiple epitopes. The “relatively short” regions arepreferably less in length than an entire native antigen. This relativelyshort sequence that contains multiple distinct or overlapping, “nested”epitopes can be used to generate a minigene construct. The construct isengineered to express the peptide, which corresponds to the nativeprotein sequence. The “relatively short” peptide is generally less than250 amino acids in length, often less than 100 amino acids in length,preferably less than 75 amino acids in length, and more preferably lessthan 50 amino acids in length. The protein sequence of the vaccinecomposition is selected because it has maximal number of epitopescontained within the sequence, i.e., it has a high concentration ofepitopes. As noted herein, epitope motifs may be nested or overlapping(i.e., frame shifted relative to one another). For example, withoverlapping epitopes, two 9-mer epitopes and one 10-mer epitope can bepresent in a 10 amino acid peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes.

The vaccine composition will include, for example, multiple CTL epitopesfrom 158P3D2 antigen and at least one HTL epitope. This polyepitopicnative sequence is administered either as a peptide or as a nucleic acidsequence which encodes the peptide. Alternatively, an analog can be madeof this native sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide.

The embodiment of this example provides for the possibility that an asyet undiscovered aspect of immune system processing will apply to thenative nested sequence and thereby facilitate the production oftherapeutic or prophylactic immune response-inducing vaccinecompositions. Additionally, such an embodiment provides for thepossibility of motif-bearing epitopes for an HLA makeup(s) that ispresently unknown. Furthermore, this embodiment (excluding an analogedembodiment) directs the immune response to multiple peptide sequencesthat are actually present in native 158P3D2, thus avoiding the need toevaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing peptide or nucleic acid vaccinecompositions.

Related to this embodiment, computer programs are available in the artwhich can be used to identify in a target sequence, the greatest numberof epitopes per sequence length.

Example 26 Polyepitopic Vaccine Compositions from Multiple Antigens

The 158P3D2 peptide epitopes of the present invention are used inconjunction with epitopes from other target tumor-associated antigens,to create a vaccine composition that is useful for the prevention ortreatment of cancer that expresses 158P3D2 and such other antigens. Forexample, a vaccine composition can be provided as a single polypeptidethat incorporates multiple epitopes from 158P3D2 as well astumor-associated antigens that are often expressed with a target cancerassociated with 158P3D2 expression, or can be administered as acomposition comprising a cocktail of one or more discrete epitopes.Alternatively, the vaccine can be administered as a minigene constructor as dendritic cells which have been loaded with the peptide epitopesin vitro.

Example 27 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response forthe presence of specific antibodies, CTL or HTL directed to 158P3D2.Such an analysis can be performed in a manner described by Ogg et al.,Science 279:2103-2106, 1998. In this Example, peptides in accordancewith the invention are used as a reagent for diagnostic or prognosticpurposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetramericcomplexes (“tetramers”) are used for a cross-sectional analysis of, forexample, 158P3D2 HLA-A*0201-specific CTL frequencies from HLAA*0201-positive individuals at different stages of disease or followingimmunization comprising a 158P3D2 peptide containing an A*0201 motif.Tetrameric complexes are synthesized as described (Musey et al., N.Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201in this example) and β2-microglobulin are synthesized by means of aprokaryotic expression system. The heavy chain is modified by deletionof the transmembrane-cytosolic tail and COOH-terminal addition of asequence containing a BirA enzymatic biotinylation site. The heavychain, β2-microglobulin, and peptide are refolded by dilution. The 45-kDrefolded product is isolated by fast protein liquid chromatography andthen biotinylated by BirA in the presence of biotin (Sigma, St. Louis,Mo.), adenosine 5′ triphosphate and magnesium.Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, andthe tetrameric product is concentrated to 1 mg/ml. The resulting productis referred to as tetramer-phycoerythrin.

For the analysis of patient blood samples, approximately one millionPBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl ofcold phosphate-buffered saline. Tri-color analysis is performed with thetetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. ThePBMCs are incubated with tetramer and antibodies on ice for 30 to 60 minand then washed twice before formaldehyde fixation. Gates are applied tocontain >99.98% of control samples. Controls for the tetramers includeboth A*0201-negative individuals and A*0201-positive non-diseaseddonors. The percentage of cells stained with the tetramer is thendetermined by flow cytometry. The results indicate the number of cellsin the PBMC sample that contain epitope-restricted CTLs, thereby readilyindicating the extent of immune response to the 158P3D2 epitope, andthus the status of exposure to 158P3D2, or exposure to a vaccine thatelicits a protective or therapeutic response.

Example 28 Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate Tcell responses, such as acute or recall responses, in patients. Such ananalysis may be performed on patients who have recovered from158P3D2-associated disease or who have been vaccinated with a 158P3D2vaccine.

For example, the class I restricted CTL response of persons who havebeen vaccinated may be analyzed. The vaccine may be any 158P3D2 vaccine.PBMC are collected from vaccinated individuals and HLA typed.Appropriate peptide epitopes of the invention that, optimally, bearsupermotifs to provide cross-reactivity with multiple HLA supertypefamily members, are then used for analysis of samples derived fromindividuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaquedensity gradients (Sigma Chemical Co., St. Louis, Mo.), washed threetimes in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCOLaboratories) supplemented with L-glutamine (2 mM), penicillin (50U/ml),streptomycin (50 μg/ml), and Hepes (10 mM) containing 10%heat-inactivated human AB serum (complete RPMI) and plated usingmicroculture formats. A synthetic peptide comprising an epitope of theinvention is added at 10 μg/ml to each well and HBV core 128-140 epitopeis added at 1 μg/ml to each well as a source of T cell help during thefirst week of stimulation.

In the microculture format, 4×10⁵ PBMC are stimulated with peptide in 8replicate cultures in 96-well round bottom plate in 100 μl/well ofcomplete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/mlfinal concentration of rIL-2 are added to each well. On day 7 thecultures are transferred into a 96-well flat-bottom plate andrestimulated with peptide, rIL-2 and 10⁵ irradiated (3,000 rad)autologous feeder cells. The cultures are tested for cytotoxic activityon day 14. A positive CTL response requires two or more of the eightreplicate cultures to display greater than 10% specific ⁵¹Cr release,based on comparison with non-diseased control subjects as previouslydescribed (Rehermann, et al., Nature Med. 2:1104, 1108, 1996; Rehermannet al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J.Clin. Invest. 98:1432-1440, 1996).

Target cell lines are autologous and allogeneic EBV-transformed B-LCLthat are either purchased from the American Society forHistocompatibility and Immunogenetics (ASHI, Boston, Mass.) orestablished from the pool of patients as described (Guilhot, et al. J.Virol. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cellsconsist of either allogeneic HLA-matched or autologous EBV-transformed Blymphoblastoid cell line that are incubated overnight with the syntheticpeptide epitope of the invention at 10 μM, and labeled with 100 μCi of⁵¹Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after whichthey are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well ⁵¹Crrelease assay using U-bottomed 96 well plates containing 3,000targets/well. Stimulated PBMC are tested at effector/target (E/T) ratiosof 20-50:1 on day 14. Percent cytotoxicity is determined from theformula: 100×[(experimental release−spontaneous release)/maximumrelease−spontaneous release)]. Maximum release is determined by lysis oftargets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis,Mo.). Spontaneous release is <25% of maximum release for allexperiments.

The results of such an analysis indicate the extent to whichHLA-restricted CTL populations have been stimulated by previous exposureto 158P3D2 or a 158P3D2 vaccine.

Similarly, Class II restricted HTL responses may also be analyzed.Purified PBMC are cultured in a 96-well flat bottom plate at a densityof 1.5×10⁵ cells/well and are stimulated with 10 μg/ml synthetic peptideof the invention, whole 158P3D2 antigen, or PHA. Cells are routinelyplated in replicates of 4-6 wells for each condition. After seven daysof culture, the medium is removed and replaced with fresh mediumcontaining 10 U/ml IL-2. Two days later, 1 μCi ³H-thymidine is added toeach well and incubation is continued for an additional 18 hours.Cellular DNA is then harvested on glass fiber mats and analyzed for³H-thymidine incorporation. Antigen-specific T cell proliferation iscalculated as the ratio of ³H-thymidine incorporation in the presence ofantigen divided by the ³H-thymidine incorporation in the absence ofantigen.

Example 29 Induction of Specific CTL Response in Humans

A human clinical trial for an immunogenic composition comprising CTL andHTL epitopes of the invention is set up as an IND Phase I, doseescalation study and carried out as a randomized, double-blind,placebo-controlled trial. Such a trial is designed, for example, asfollows:

A total of about 27 individuals are enrolled and divided into 3 groups:

-   -   Group I: 3 subjects are injected with placebo and 6 subjects are        injected with 5 pg of peptide composition;    -   Group II: 3 subjects are injected with placebo and 6 subjects        are injected with 50 μg peptide composition;    -   Group III: 3 subjects are injected with placebo and 6 subjects        are injected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive abooster inoculation at the same dosage.

The endpoints measured in this study relate to the safety andtolerability of the peptide composition as well as its immunogenicity.Cellular immune responses to the peptide composition are an index of theintrinsic activity of this the peptide composition, and can therefore beviewed as a measure of biological efficacy. The following summarize theclinical and laboratory data that relate to safety and efficacyendpoints.

Safety: The incidence of adverse events is monitored in the placebo anddrug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy,subjects are bled before and after injection. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

The vaccine is found to be both safe and efficacious.

Example 30 Phase II Trials In Patients Expressing 158P3D2

Phase II trials are performed to study the effect of administering theCTL-HTL peptide compositions to patients having cancer that expresses158P3D2. The main objectives of the trial are to determine an effectivedose and regimen for inducing CTLs in cancer patients that express158P3D2, to establish the safety of inducing a CTL and HTL response inthese patients, and to see to what extent activation of CTLs improvesthe clinical picture of these patients, as manifested, e.g., by thereduction and/or shrinking of lesions. Such a study is designed, forexample, as follows:

The studies are performed in multiple centers. The trial design is anopen-label, uncontrolled, dose escalation protocol wherein the peptidecomposition is administered as a single dose followed six weeks later bya single booster shot of the same dose. The dosages are 50, 500 and5,000 micrograms per injection. Drug-associated adverse effects(severity and reversibility) are recorded.

There are three patient groupings. The first group is injected with 50micrograms of the peptide composition and the second and third groupswith 500 and 5,000 micrograms of peptide composition, respectively. Thepatients within each group range in age from 21-65 and represent diverseethnic backgrounds. All of them have a tumor that expresses 158P3D2.

Clinical manifestations or antigen-specific T-cell responses aremonitored to assess the effects of administering the peptidecompositions. The vaccine composition is found to be both safe andefficacious in the treatment of 158P3D2-associated disease.

Example 31 Induction of CTL Responses Using a Prime Boost Protocol

A prime boost protocol similar in its underlying principle to that usedto confirm the efficacy of a DNA vaccine in transgenic mice, such asdescribed above in the Example entitled “The Plasmid Construct and theDegree to Which It Induces Immunogenicity,” can also be used for theadministration of the vaccine to humans. Such a vaccine regimen caninclude an initial administration of, for example, naked DNA followed bya boost using recombinant virus encoding the vaccine, or recombinantprotein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization may be performed using anexpression vector, such as that constructed in the Example entitled“Construction of “Minigene” Multi-Epitope DNA Plasmids” in the form ofnaked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also beadministered using a gene gun. Following an incubation period of 3-4weeks, a booster dose is then administered. The booster can berecombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu.An alternative recombinant virus, such as an MVA, canarypox, adenovirus,or adeno-associated virus, can also be used for the booster, or thepolyepitopic protein or a mixture of the peptides can be administered.For evaluation of vaccine efficacy, patient blood samples are obtainedbefore immunization as well as at intervals following administration ofthe initial vaccine and booster doses of the vaccine. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

Analysis of the results indicates that a magnitude of responsesufficient to achieve a therapeutic or protective immunity against158P3D2 is generated.

Example 32 Administration of Vaccine Compositions Using Dendritic Cells(DC)

Vaccines comprising peptide epitopes of the invention can beadministered using APCs, or “professional” APCs such as DC. In thisexample, peptide-pulsed DC are administered to a patient to stimulate aCTL response in vivo. In this method, dendritic cells are isolated,expanded, and pulsed with a vaccine comprising peptide CTL and HTLepitopes of the invention. The dendritic cells are infused back into thepatient to elicit CTL and HTL responses in vivo. The induced CTL and HTLthen destroy or facilitate destruction, respectively, of the targetcells that bear the 158P3D2 protein from which the epitopes in thevaccine are derived.

For example, a cocktail of epitope-comprising peptides is administeredex vivo to PBMC, or isolated DC therefrom. A pharmaceutical tofacilitate harvesting of DC can be used, such as Progenipoietin™(Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC withpeptides, and prior to reinfusion into patients, the DC are washed toremove unbound peptides.

As appreciated clinically, and readily determined by one of skill basedon clinical outcomes, the number of DC reinfused into the patient canvary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 andProstate 32:272, 1997). Although 2-50×10⁶ DC per patient are typicallyadministered, larger number of DC, such as 10⁷ or 10⁸ can also beprovided. Such cell populations typically contain between 50-90% DC.

In some embodiments, peptide-loaded PBMC are injected into patientswithout purification of the DC. For example, PBMC generated aftertreatment with an agent such as Progenipoietin™ are injected intopatients without purification of the DC. The total number of PBMC thatare administered often ranges from 10⁸ to 10¹⁰. Generally, the celldoses injected into patients is based on the percentage of DC in theblood of each patient, as determined, for example, by immunofluorescenceanalysis with specific anti-DC antibodies. Thus, for example, ifProgenipoietin™ mobilizes 2% DC in the peripheral blood of a givenpatient, and that patient is to receive 5×10⁶ DC, then the patient willbe injected with a total of 2.5×10⁸ peptide-loaded PBMC. The percent DCmobilized by an agent such as Progenipoietin™ is typically estimated tobe between 2-10%, but can vary as appreciated by one of skill in theart.

Ex Vivo Activation of CTL/HTL Responses

Alternatively, ex vivo CTL or HTL responses to 158P3D2 antigens can beinduced by incubating, in tissue culture, the patient's, or geneticallycompatible, CTL or HTL precursor cells together with a source of APC,such as DC, and immunogenic peptides. After an appropriate incubationtime (typically about 7-28 days), in which the precursor cells areactivated and expanded into effector cells, the cells are infused intothe patient, where they will destroy (CTL) or facilitate destruction(HTL) of their specific target cells, i.e., tumor cells.

Example 33 An Alternative Method of Identifying and ConfirmingMotif-Bearing Peptides

Another method of identifying and confirming motif-bearing peptides isto elute them from cells bearing defined MHC molecules. For example, EBVtransformed B cell lines used for tissue typing have been extensivelycharacterized to determine which HLA molecules they express. In certaincases these cells express only a single type of HLA molecule. Thesecells can be transfected with nucleic acids that express the antigen ofinterest, e.g. 158P3D2. Peptides produced by endogenous antigenprocessing of peptides produced as a result of transfection will thenbind to HLA molecules within the cell and be transported and displayedon the cell's surface. Peptides are then eluted from the HLA moleculesby exposure to mild acid conditions and their amino acid sequencedetermined, e.g., by mass spectral analysis (e.g., Kubo et al., J.Immunol. 152:3913, 1994). Because the majority of peptides that bind aparticular HLA molecule are motif-bearing, this is an alternativemodality for obtaining the motif-bearing peptides correlated with theparticular HLA molecule expressed on the cell.

Alternatively, cell lines that do not express endogenous HLA moleculescan be transfected with an expression construct encoding a single HLAallele. These cells can then be used as described, i.e., they can thenbe transfected with nucleic acids that encode 158P3D2 to isolatepeptides corresponding to 158P3D2 that have been presented on the cellsurface. Peptides obtained from such an analysis will bear motif(s) thatcorrespond to binding to the single HLA allele that is expressed in thecell.

As appreciated by one in the art, one can perform a similar analysis ona cell bearing more than one HLA allele and subsequently determinepeptides specific for each HLA allele expressed. Moreover, one of skillwould also recognize that means other than transfection, such as loadingwith a protein antigen, can be used to provide a source of antigen tothe cell.

Example 34 Complementary Polynucleotides

Sequences complementary to the 158P3D2-encoding sequences or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring 158P3D2. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06software (National Biosciences) and the coding sequence of 158P3D2. Toinhibit transcription, a complementary oligonucleotide is designed fromthe most unique 5′ sequence and used to prevent promoter binding to thecoding sequence. To inhibit translation, a complementary oligonucleotideis designed to prevent ribosomal binding to a 158P3D2-encodingtranscript.

Example 35 Purification of Naturally-Occurring or Recombinant 158P3D2Using 158P3D2-Specific Antibodies

Naturally occurring or recombinant 158P3D2 is substantially purified byimmunoaffinity chromatography using antibodies specific for 158P3D2. Animmunoaffinity column is constructed by covalently coupling anti-158P3D2antibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing 158P3D2 are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of 158P3D2 (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/158P3D2 binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andGCR.P is collected.

Example 36 Identification of Molecules which Interact with 158P3D2

158P3D2, or biologically active fragments thereof, are labeled with 1211 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled 158P3D2, washed, and anywells with labeled 158P3D2 complex are assayed. Data obtained usingdifferent concentrations of 158P3D2 are used to calculate values for thenumber, affinity, and association of 158P3D2 with the candidatemolecules.

Example 37 In Vivo Assay for 158P3D2 Tumor Growth Promotion

In Vivo Assay of 3T3 Cell Growth by Recombinant Expression of 158P3D2.

To address the determination of 158P3D2 to accelerate the growth ofnon-tumorigenic cells in an in vivo mouse model, non-transformed 3T3cells are prepared by infection with either a virus containing an emptyvector control (Neo gene alone) or with a vector containing the 158P3D2full-length gene. 3T3 cells are selected for survival in G-418, andexpression of 158P3D2 confirmed by Northern blot analysis. To assess thegrowth of these cells, 1×10⁶ 158P3D2 expressing 3T3 cells or 1×10⁶ Neocontrol are mixed with Matrigel®, then injected intratibially orsubcutaneously in SCID mice and allowed to grow for 30 days. The growthof these cells is assessed on day 30 by visual inspection and bynecropsy. The 158P3D2 expressing 3T3 cells show a potent effect incomparison to the 3T3-Neo cells, indicating that the 158P3D2 proteinenhanced the growth of the cells in Matrigel®. 158P3D2 promotes thegrowth of non-tumorigenic cells and provides a growth advantage in vivothat mimics the role of this protein in human malignancies.

Example 38 158P3D2 Monoclonal Antibody-Mediated Inhibition of Bladder,Lung Colon and Breast and other Tumors In Vivo

The significant expression of 158P3D2 in cancer tissues, together withits restrictive expression in normal tissues makes 158P3D2 a good targetfor antibody therapy. Similarly, 158P3D2 is a target for T cell-basedimmunotherapy. Thus, the therapeutic efficacy of anti-158P3D2 MAbs inhuman bladder cancer xenograft mouse models is evaluated by usingrecombinant cell lines such as J82-158P3D2 (see, e.g., Kaighn, M. E., etal., Invest Urol, 1979. 17(1): p. 16-23), as well as human bladderxenograft models (SCaBER).

Antibody efficacy on tumor growth and metastasis formation is studied,e.g., in a mouse orthotopic bladder cancer xenograft model. Theantibodies can be unconjugated, as discussed in this Example, or can beconjugated to a therapeutic modality (see below), as appreciated in theart. Anti-158P3D2 MAbs inhibit formation of bladder xenografts.Anti-158P3D2 MAbs retard the growth of established orthotopic tumors andprolong survival of tumor-bearing mice. MAb effects on tumor growth inmouse models support the utility of anti-158P3D2 MAbs in the treatmentof local and advanced stages of bladder cancer (see, e.g., Saffran, D.,2001, et al., PNAS 10:1073-1078).

Administration of the anti-158P3D2 MAbs leads to retardation ofestablished orthotopic tumor growth and inhibition of metastasis todistant sites, resulting in a significant prolongation in the survivalof tumor-bearing mice. Therefore, 158P3D2 is an attractive target forimmunotherapy, and anti-158P3D2 MAbs have therapeutic potential for thetreatment of local and metastatic cancer. This example demonstrates thatunconjugated 158P3D2 monoclonal antibodies are effective to inhibit thegrowth of human bladder tumor xenografts grown in SCID mice;accordingly, a combination of such efficacious MAbs is also effective.

MAb-Toxin Conjugates:

Another embodiment of MAb therapy is through the use of toxinconjugation of MAbs for targeted delivery of cytotoxic agents to cellsexpressing the protein target. Major advances have been made in theclinical application of MAb toxin conjugates with the development ofMylotarg for acute myeloid leukemia (Bross, P. F., et al., 2001, Clin.Cancer Res. 7:1490-1496). Mylotarg is a humanized MAb directed to CD33which is conjugated to a highly potent DNA-alkylating agent(calichemicin) via an acid labile hydrazone bond (Hamann, P. R., et al.,2002, Bioconjug. Chem. 13:40-46; ibid., 13:47-58). Additional toxins forMAb conjugation in development include maytansinoid, doxorubicin,taxoids and the potent synthetic dolastatin 10 analogs auristatin E andmonomethylauristatin E (Doronina, S. O., et al., 2003, Nature Biotech.21:778-784; Ross, S., et al., 2002, Cancer Res. 62:2546-2553; Francisco,J. A., et el., 2003, Blood 102: 1458-1465; Mao, W., et al., 2004, CancerRes. 64:781-788). Such applications have potential to deliver acytotoxic agent to cells expressing the protein target of the MAb.Internalization of the target protein upon MAb binding is important fortoxin delivery, and the mechanism spares the non-targeted tissues fromthe potentially harmful effects of the cytotoxic agent.

158P3D2 MAbs conjugated to toxins are used to induce cell killing invitro using established protocols for cytotoxicity assays and clonogenicassays (Doronina, S. O., et al., 2003, Nature Biotech. 21:778-784; Mao,W., et al., 2004, Cancer Res. 64:781-788). Toxin conjugated anti-158P3D2MAbs induce cytotoxicity of cells expressing endogenous 158P3D2 (SCaBERcells) and recombinant 158P3D2 (PC3-158P3D2, 3T3-158P3D2, Rat-1-158P3D2and B300.19-158P3D2). This methodology allows confirmation that thetoxin conjugated MAb is functional against cells expressing the 158P3D2protein on their surface versus those that do not express the target.

The MAb toxin conjugates are tested for their ability to inhibit tumorgrowth in vivo. Antibody efficacy on tumor growth and metastasisformation is studied, e.g., in a mouse orthotopic bladder cancerxenograft model, a mouse lung cancer xenograft model, or mouse colon orbreast cancer xenograft model. Administration of the anti-158P3D2 MAbsled to retardation of established orthotopic tumor growth and inhibitionof metastasis to distant sites, resulting in a significant prolongationin the survival of tumor-bearing mice. These studies indicate that158P3D2 is an attractive target for immunotherapy and demonstrate thetherapeutic potential of toxin-conjugated anti-158P3D2 MAbs for thetreatment of local and metastatic cancer. This example demonstrates thattoxin-conjugated 158P3D2 monoclonal antibodies are effective to inhibitthe growth of human bladder, lung, breast and colon tumor xenograftsgrown in SCID mice; accordingly, a combination of such efficacious MAbsis also effective. The methodology allows the targeted delivery of acytotoxin using a plasma stable linker in a MAb-toxin conjugate. Such amechanism of action reduces the potential harmful effects of the toxinon non-targeted tissues.

Tumor Inhibition Using Multiple Unconjugated or Toxin-Conjugated 158P3D2MAbs

Materials and Methods

158P3D2 Monoclonal Antibodies:

Monoclonal antibodies were raised against 158P3D2 as described in theExample entitled “Generation of 158P3D2 Monoclonal Antibodies (MAbs).”The antibodies are characterized by ELISA, Western blot, FACS, andimmunoprecipitation for their capacity to bind 158P3D2. Epitope mappingdata for the anti-158P3D2 MAbs, as determined by ELISA and Westernanalysis, recognize epitopes on the 158P3D2 protein. Immunohistochemicalanalysis of bladder cancer tissues and cells with these antibodies isperformed.

The monoclonal antibodies are purified from ascites or hybridoma tissueculture supernatants by Protein-G Sepharose chromatography, dialyzedagainst PBS, filter sterilized, and stored at −20° C. Proteindeterminations are performed by a Bradford assay (Bio-Rad, Hercules,Calif.). A therapeutic monoclonal antibody or a cocktail comprising amixture of individual monoclonal antibodies is prepared and used for thetreatment of mice receiving subcutaneous or orthotopic injections ofSCaBER or J82-158P3D2 tumor xenografts.

The MAbs to 158P3D2 are conjugated to various different toxins (listedabove) using any of a variety of methods described elsewhere in the art(Hamann, P. R., et al., 2002, Bioconjug. Chem. 13:40-46; ibid.,13:47-58; Doronina, S. O., et al., 2003, Nature Biotech. 21:778-784;Ojima, I., et al. 2002, J. Med. Chem. 45:5620-5623; Dubowchik, G. M., etal., 2002, Bioconjug. Chem. 13:855-869; King, H. D., 2002, J. Med. Chem45:4336-4343; Ross, S., et al., 2002, Cancer Res. 62:2546-2553;Francisco, J. A., et el., 2003, Blood 102:1458-1465 Mao, W., et al.,2004, Cancer Res. 64:781-788).

Cell Lines

The bladder carcinoma cell lines, J82 and SCaBER, as well as thefibroblast line NIH 3T3 (American Type Culture Collection) aremaintained in media supplemented with L-glutamine and 10% FBS.J82-158P3D2 and 3T3-158P3D2 cell populations are generated by retroviralgene transfer as described in Hubert, R. S., et al., Proc. Natl. Acad.Sci. USA, 1999, 96(25): 14523.

Xenograft Mouse Models

Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶ cancercells mixed at a 1:1 dilution with Matrigel® (Collaborative Research) inthe right flank of male SCID mice. To test antibody efficacy on tumorformation, i.p. antibody injections are started on the same day astumor-cell injections. As a control, mice are injected with eitherpurified mouse IgG (ICN) or PBS; or a purified monoclonal antibody thatrecognizes an irrelevant protein not expressed in human cells. Tumorsizes are determined by caliper measurements, and the tumor volume iscalculated as: Length×Width×Height. Mice with s.c. tumors greater than1.5 cm in diameter are sacrificed.

Orthotopic injections are performed under anesthesia by usingketamine/xylazine. For bladder orthotopic studies, an incision is madethrough the abdomen to expose the bladder, and tumor cells (5×10⁵) mixedwith Matrigel® are injected into the bladder wall in a 10-μl volume. Tomonitor tumor growth, mice are palpated and blood is collected on aweekly basis to measure BTA levels. For prostate orthopotic models, anincision is made through the abdominal muscles to expose the bladder andseminal vesicles, which then are delivered through the incision toexpose the dorsal prostate. Tumor cells, e.g. SCaBER cells (5×10⁵) mixedwith Matrigel® are injected into the bladder in a 10-μl volume (YoshidaY et al, Anticancer Res. 1998, 18:327; Ahn et al, Tumor Biol. 2001,22:146). The mice are segregated into groups for the appropriatetreatments, with anti-158P3D2 or control MAbs being injected i.p.

Anti-158P3D2 MAbs Inhibit Growth of 158P3D2-Expressing Xenograft-CancerTumors

The effect of anti-158P3D2 MAbs on tumor formation is tested on thegrowth and progression of bladder cancer xenografts using SCaBER andJ82-158P3D2 orthotopic models. As compared with the s.c. tumor model,the orthotopic model, which requires injection of tumor cells directlyin the mouse bladder, and prostate, respectively, results in a localtumor growth, development of metastasis in distal sites, deteriorationof mouse health, and subsequent death (Saffran, D., et al., PNAS supra;Fu, X., et al., Int J Cancer, 1992. 52(6): p. 987-90; Kubota, T., J CellBiochem., 1994. 56(1): p. 4-8). The features make the orthotopic modelmore representative of human disease progression and allowed us tofollow the therapeutic effect of MAbs on clinically relevant end points.

Accordingly, tumor cells are injected into the mouse bladder, or lung,and 2 days later, the mice are segregated into two groups and treatedwith either: a) 200-500 μg of anti-158P3D2 MAb, or b) PBS three timesper week for two to five weeks.

A major advantage of the orthotopic cancer models is the ability tostudy the development of metastases. Formation of metastasis in micebearing established orthotopic tumors is studies by IHC analysis on lungsections using an antibody against a tumor-specific cell-surface proteinsuch as anti-cytokeratin 20 for bladder cancer models (Lin S et al,Cancer Detect Prev. 2001;25:202).

Mice bearing established orthotopic tumors are administered 1000 μginjections of either anti-158P3D2 MAb or PBS over a 4-week period. Micein both groups are allowed to establish a high tumor burden, to ensure ahigh frequency of metastasis formation in mouse lungs. Mice then arekilled and their bladders, livers, bone and lungs are analyzed for thepresence of tumor cells by IHC analysis.

Anti-158P3D2 antibodies inhibit the formation of tumors, retard thegrowth of already established tumors, and prolong the survival oftreated mice. Moreover, anti-158P3D2 MAbs demonstrate a dramaticinhibitory effect on the spread of local bladder tumors to distal sites,even in the presence of a large tumor burden. Thus, anti-158P3D2 MAbsare efficacious on major clinically relevant end points (tumor growth),prolongation of survival, and health.

Example 39 Therapeutic and Diagnostic Use of Anti-158P3D2 Antibodies inHumans

Anti-158P3D2 monoclonal antibodies are safely and effectively used fordiagnostic, prophylactic, prognostic and/or therapeutic purposes inhumans. Western blot and immunohistochemical analysis of cancer tissuesand cancer xenografts with anti-158P3D2 mAb show strong extensivestaining in carcinoma but significantly lower or undetectable levels innormal tissues. Detection of 158P3D2 in carcinoma and in metastaticdisease demonstrates the usefulness of the mAb as a diagnostic and/orprognostic indicator. Anti-158P3D2 antibodies are therefore used indiagnostic applications such as immunohistochemistry of kidney biopsyspecimens to detect cancer from suspect patients.

As determined by flow cytometry, anti-158P3D2 mAb specifically binds tocarcinoma cells. Thus, anti-158P3D2 antibodies are used in diagnosticwhole body imaging applications, such as radioimmunoscintigraphy andradioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res20(2A):925-948 (2000)) for the detection of localized and metastaticcancers that exhibit expression of 158P3D2. Shedding or release of anextracellular domain of 158P3D2 into the extracellular milieu, such asthat seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology27:563-568 (1998)), allows diagnostic detection of 158P3D2 byanti-158P3D2 antibodies in serum and/or urine samples from suspectpatients.

Anti-158P3D2 antibodies that specifically bind 158P3D2 are used intherapeutic applications for the treatment of cancers that express158P3D2. Anti-158P3D2 antibodies are used as an unconjugated modalityand as conjugated form in which the antibodies are attached to one ofvarious therapeutic or imaging modalities well known in the art, such asa prodrugs, enzymes or radioisotopes. In preclinical studies,unconjugated and conjugated anti-158P3D2 antibodies are tested forefficacy of tumor prevention and growth inhibition in the SCID mousecancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6,(see, e.g., the Example entitled “158P3D2 Monoclonal Antibody-mediatedInhibition of Bladder and Lung Tumors In Vivo”). Either conjugated andunconjugated anti-158P3D2 antibodies are used as a therapeutic modalityin human clinical trials either alone or in combination with othertreatments as described in following Examples.

Example 40 Human Clinical Trials for the Treatment and Diagnosis ofHuman Carcinomas Through Use of Human Anti-158P3D2 Antibodies In Vivo

Antibodies are used in accordance with the present invention whichrecognize an epitope on 158P3D2, and are used in the treatment ofcertain tumors such as those listed in Table I. Based upon a number offactors, including 158P3D2 expression levels, tumors such as thoselisted in Table I are presently preferred indications. In connectionwith each of these indications, three clinical approaches aresuccessfully pursued.

Adjunctive therapy: In adjunctive therapy, patients are treated withanti-158P3D2 antibodies in combination with a chemotherapeutic orantineoplastic agent and/or radiation therapy. Primary cancer targets,such as those listed in Table I, are treated under standard protocols bythe addition anti-158P3D2 antibodies to standard first and second linetherapy. Protocol designs address effectiveness as assessed by reductionin tumor mass as well as the ability to reduce usual doses of standardchemotherapy. These dosage reductions allow additional and/or prolongedtherapy by reducing dose-related toxicity of the chemotherapeutic agent.Anti-158P3D2 antibodies are utilized in several adjunctive clinicaltrials in combination with the chemotherapeutic or antineoplastic agentsadriamycin (advanced prostrate carcinoma), cisplatin (advanced head andneck and lung carcinomas), taxol (breast cancer), and doxorubicin(preclinical).

Monotherapy: In connection with the use of the anti-158P3D2 antibodiesin monotherapy of tumors, the antibodies are administered to patientswithout a chemotherapeutic or antineoplastic agent. In one embodiment,monotherapy is conducted clinically in end stage cancer patients withextensive metastatic disease. Patients show some disease stabilization.Trials demonstrate an effect in refractory patients with canceroustumors.

Imaging Agent: Through binding a radionuclide (e.g., iodine or yttrium(I¹³¹, Y⁹⁰) to anti-158P3D2 antibodies, the radiolabeled antibodies areutilized as a diagnostic and/or imaging agent. In such a role, thelabeled antibodies localize to both solid tumors, as well as, metastaticlesions of cells expressing 158P3D2. In connection with the use of theanti-158P3D2 antibodies as imaging agents, the antibodies are used as anadjunct to surgical treatment of solid tumors, as both a pre-surgicalscreen as well as a post-operative follow-up to determine what tumorremains and/or returns. In one embodiment, a (¹¹¹In)-158P3D2 antibody isused as an imaging agent in a Phase I human clinical trial in patientshaving a carcinoma that expresses 158P3D2 (by analogy see, e.g., Divgiet al. J. Natl. Cancer Inst. 83:97-104 (1991)). Patients are followedwith standard anterior and posterior gamma camera. The results indicatethat primary lesions and metastatic lesions are identified.

Dose and Route of Administration

As appreciated by those of ordinary skill in the art, dosingconsiderations can be determined through comparison with the analogousproducts that are in the clinic. Thus, anti-158P3D2 antibodies can beadministered with doses in the range of 5 to 400 mg/m², with the lowerdoses used, e.g., in connection with safety studies. The affinity ofanti-158P3D2 antibodies relative to the affinity of a known antibody forits target is one parameter used by those of skill in the art fordetermining analogous dose regimens. Further, anti-158P3D2 antibodiesthat are fully human antibodies, as compared to the chimeric antibody,have slower clearance; accordingly, dosing in patients with such fullyhuman anti-158P3D2 antibodies can be lower, perhaps in the range of 50to 300 mg/m², and still remain efficacious. Dosing in mg/m², as opposedto the conventional measurement of dose in mg/kg, is a measurement basedon surface area and is a convenient dosing measurement that is designedto include patients of all sizes from infants to adults.

Three distinct delivery approaches are useful for delivery ofanti-158P3D2 antibodies. Conventional intravenous delivery is onestandard delivery technique for many tumors. However, in connection withtumors in the peritoneal cavity, such as tumors of the ovaries, biliaryduct, other ducts, and the like, intraperitoneal administration mayprove favorable for obtaining high dose of antibody at the tumor and toalso minimize antibody clearance. In a similar manner, certain solidtumors possess vasculature that is appropriate for regional perfusion.Regional perfusion allows for a high dose of antibody at the site of atumor and minimizes short term clearance of the antibody.

Clinical Development Plan (CDP)

Overview: The CDP follows and develops treatments of anti-158P3D2antibodies in connection with adjunctive therapy, monotherapy, and as animaging agent. Trials initially demonstrate safety and thereafterconfirm efficacy in repeat doses. Trails are open label comparingstandard chemotherapy with standard therapy plus anti-158P3D2antibodies. As will be appreciated, one criteria that can be utilized inconnection with enrollment of patients is 158P3D2 expression levels intheir tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safetyconcerns are related primarily to (i) cytokine release syndrome, i.e.,hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 158P3D2.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Anti-158P3D2 antibodies are found to be safe upon humanadministration.

Example 41 Human Clinical Trial Adjunctive Therapy with HumanAnti-158P3D2 Antibody and Chemotherapeutic Agent

A phase I human clinical trial is initiated to assess the safety of sixintravenous doses of a human anti-158P3D2 antibody in connection withthe treatment of a solid tumor, e.g., a cancer of a tissue listed inTable I. In the study, the safety of single doses of anti-158P3D2antibodies when utilized as an adjunctive therapy to an antineoplasticor chemotherapeutic agent as defined herein, such as, withoutlimitation: cisplatin, topotecan, doxorubicin, adriamycin, taxol, or thelike, is assessed. The trial design includes delivery of six singledoses of an anti-158P3D2 antibody with dosage of antibody escalatingfrom approximately about 25 mg/m ² to about 275 mg/m ² over the courseof the treatment in accordance with the following schedule: Day 0 Day 7Day 14 Day 21 Day 28 Day 35 mAb Dose 25 mg/m² 75 mg/m² 125 mg/m² 175mg/m² 225 mg/m² 275 mg/m² Chemotherapy + + + + + + (standard dose)

Patients are closely followed for one-week following each administrationof antibody and chemotherapy. In particular, patients are assessed forthe safety concerns mentioned above: (i) cytokine release syndrome,i.e., hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the human antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 158P3D2.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Patients are also assessed for clinical outcome, andparticularly reduction in tumor mass as evidenced by MRI or otherimaging.

The anti-158P3D2 antibodies are demonstrated to be safe and efficacious,Phase II trials confirm the efficacy and refine optimum dosing.

Example 42 Human Clinical Trial: Monotherapy with Human Anti-158P3D2Antibody

Anti-158P3D2 antibodies are safe in connection with the above-discussedadjunctive trial, a Phase II human clinical trial confirms the efficacyand optimum dosing for monotherapy. Such trial is accomplished, andentails the same safety and outcome analyses, to the above-describedadjunctive trial with the exception being that patients do not receivechemotherapy concurrently with the receipt of doses of anti-158P3D2antibodies.

Example 43 Human Clinical Trial: Diagnostic Imaging with Anti-158P3D2Antibody

Once again, as the adjunctive therapy discussed above is safe within thesafety criteria discussed above, a human clinical trial is conductedconcerning the use of anti-158P3D2 antibodies as a diagnostic imagingagent. The protocol is designed in a substantially similar manner tothose described in the art, such as in Divgi et al. J. Natl. CancerInst. 83:97-104 (1991). The antibodies are found to be both safe andefficacious when used as a diagnostic modality.

Example 44 158P3D2 Functional Assays

158P3D2 protein, and variants thereof, is a member of a family ofrelated proteins, the ferlins. This family of membrane proteins ischaracterized by the presence of intracellular C2 domains, so named bytheir homology to a conserved protein kinase C (PKC) motif. Thecanonical C2 domain is a 130 amino acid long Ca²⁺ dependent membranetargeting module that is found in proteins involved in signaltransduction or membrane trafficking (Rizo, J. and Sudhof, T. C., J.Biol. Chem. 273, 15879-82 (1998)). The function of the C2 domain amongstthe >100 proteins identified to date varies between these proteins,however a common feature is that the C2 domain has been shown to bind tophospholipids, particularly phosphatidylserine and phosphatidylcholine.In some cases, the C2 domain may not bind to Ca²⁺ or to phospholipidsbut rather to other proteins (Rizo, J. and Sudhof, T. C., J. Biol. Chem.273, 15879-82 (1998)). 158P3D2, and variants thereof, are Ca²⁺ bindingproteins with the capacity to bind to both phospholipids and toproteins. The different variants of 158P3D2, which express differentnumbers of C2 domains, have different functions with respect to theunique combinations of expressed C2 regions.

Dysferlin is a member of this family of C2 containing proteins that hasa function in muscle membrane repair. Human mutation of dysferlin leadsto specific autosomal recessive muscular dystrophies (limb-girdle MDtype 2B and Miyoshi myopathy) (reviewed in Bansal, D. and Campbell, K.P., Trends in Cell Biol. 14, 206-213). Dysferlin is localized in theplasma membrane of cells where it interacts with annexin A1 and A2, andis also found in vesicles. Membrane disruption (for example in muscle)causes an increase of localized Ca²⁺ at the wound site and anaccumulation of vesicles containing dysferlin. The dysferlin proteinfacilitates both docking and fusion of the vesicles with the plasmamembrane through interaction with the annexins and/or othermembrane-associated proteins. Fusion between the repair vesicles and theplasma membrane seals the wound (Bansal, D. and Campbell, K. P., Trendsin Cell Biol. 14, 206-213).

158P3D2 protein, and variants thereof, functions in a similar fashion asdysferlin by inducing repair of cellular plasma membranes followingtheir disruption. Given the high rate of cell division and stressconditions such as hypoxia and reduced nutrient supply during tumorformation, membrane repair becomes a critical component of tumorsurvival. Expression of 158P3D2, and variants thereof, on tumorsprovides an advantage for such cells to grow under stressful conditionssuch as hypoxia or nutrient deprivation.

The C2 domain of the lipid phosphatase/tumor suppressor PTEN isregulated by threonine phosphorylation (Raftopolou, M., et al., 2004,Science, 303, 1179-81). This phosphorylation event inhibits cellmigration independent of the lipid phosphatase activity, which mayrelate to the tumor suppressive activity of PTEN. However, given theregulation of C2-induced function by phosphorylation, the status of thatphosphorylation event alters the migratory capacity of the cell. 158P3D2protein, and variants thereof, reside in the plasma membrane of tumorcells as C2-containing regulators of cell migration due to alterationsin the phosphorylation status of 158P3D2. Upon phosphorylation of158P3D2, the C2 domains influence the migratory capacity of158P3D2-positive tumor cells, conferring an advantage for them tomigrate to distal sites to seek secondary growth (metastasis). 158P3D2,and variants thereof, also bind to signal transduction proteins,providing important signaling cascades for tumor cells that confer agrowth advantage and increased capacity for cell migration and adhesion.Such advantages are key elements for increased survival and metastasisfor bladder, lung, colon and breast cancer cells.

Enhanced proliferation and entry into S-phase of tumor cells relative tonormal cells is a hallmark of the cancer cell phenotype. To address theeffect of expression of 158P3D2 on the proliferation rate of normalcells, two rodent cell lines (3T3 and Rat-1) are infected with viruscontaining the 158P3D2 gene and stable cells expressing 158P3D2 antigenare derived, as well as empty vector control cells expressing theselection marker neomycin (Neo). The cells are grown overnight in 0.5%FBS and then compared to cells treated with 10% FBS. The cells areevaluated for proliferation at 18-96 hr post-treatment by a ³H-thymidineincorporation assay and for cell cycle analysis by a BrdUincorporation/propidium iodide staining assay. Rat-1 cells expressingthe 158P3D2 antigen grow effectively in low serum concentrations (0.1%)compared to the Rat-1-Neo cells. Similar data are obtained for the 3T3cells expressing 158P3D2 versus Neo only. To assess cell proliferationby another methodology, the cells are stained with BrdU and propidiumiodide. Briefly, cells are labeled with 10 □M BrdU, washed, trypsinizedand fixed in 0.4% paraformaldehyde and 70% ethanol. Anti-BrdU-FITC(Pharmigen) is added to the cells, the cells are washed and thenincubated with 10 □g/ml propidium iodide for 20 min prior to washing andanalysis for fluorescence at 488 nm. An increase in labeling of cells inS-phase (DNA synthesis phase of the cell cycle) in 3T3 cells thatexpress the 158P3D2 protein is observed relative to control cells. Thisconfirms the results of those measured by ³H-thymidine incorporation.Accordingly, 158P3D2 expressing cells have increased potential forgrowth as tumor cells in vivo during stress, including nutrientdeprivation, hypoxia or reduced osmolarity.

Example 45 158P3D2 RNA Interference (RNAi)

RNA interference (RNAi) technology is implemented to a variety of cellassays relevant to oncology. RNAi is a post-transcriptional genesilencing mechanism activated by double-stranded RNA (dsRNA). RNAiinduces specific mRNA degradation leading to changes in proteinexpression and subsequently in gene function. In mammalian cells, thesedsRNAs called short interfering RNA (siRNA) have the correct compositionto activate the RNAi pathway targeting for degradation, specificallysome mRNAs. See, Elbashir S. M., et al., Duplexes of 21-nucleotide RNAsMediate RNA interference in Cultured Mammalian Cells, Nature411(6836):494-8 (2001). Thus, RNAi technology is used successfully inmammalian cells to silence targeted genes.

Loss of cell proliferation control is a hallmark of cancerous cells;thus, assessing the role of 158P3D2 in cell survival/proliferationassays is relevant. Accordingly, RNAi was used to investigate thefunction of the 158P3D2 antigen. To generate siRNA for 158P3D2,algorithms were used that predict oligonucleotides that exhibit thecritical molecular parameters (G:C content, melting temperature, etc.)and have the ability to significantly reduce the expression levels ofthe 158P3D2 protein when introduced into cells. Accordingly, onetargeted sequence for the 158P3D2 siRNA is: 5′CCTCGGCAGCCAATCAGCTAT 3′(SEQ ID NO: 104) (oligo 158P3D2.b). In accordance with this Example,158P3D2 siRNA compositions are used that comprise siRNA (doublestranded, short interfering RNA) that correspond to the nucleic acid ORFsequence of the 158P3D2 protein or subsequences thereof. Thus, siRNAsubsequences are used in this manner are generally 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35 or more than 35 contiguous RNA nucleotides inlength. These siRNA sequences are complementary and non-complementary toat least a portion of the mRNA coding sequence. In a preferredembodiment, the subsequences are 19-25 nucleotides in length, mostpreferably 21-23 nucleotides in length. In preferred embodiments, thesesiRNA achieve knockdown of 158P3D2 antigen in cells expressing theprotein and have functional effects as described below.

The selected siRNA (158P3D2.b oligo) was tested in numerous cell linesin the thymidine incorporation/proliferation assay (measures ³H-Thyuptake and incorporation into DNA). Moreover, this 158P3D2.b oligoachieved knockdown of 158P3D2 antigen in cells expressing the proteinand had functional effects as described below using the followingprotocols.

Mammalian siRNA transfections: The day before siRNA transfection, thedifferent cell lines were plated in media (RPMI 1640 with 10% FBS w/oantibiotics) at 2×10³ cells/well in 80 □l (96 well plate format) for thesurvival/MTS assay. In parallel with the 158P3D2 specific siRNA oligo,the following sequences were included in every experiment as controls:a) Mock transfected cells with Lipofectamine 2000 (Invitrogen, Carlsbad,Calif.) and annealing buffer (no siRNA); b) Luciferase-4 specific siRNA(targeted sequence: 5′-AAGGGACGAAGACGAACACUUCTT-3′) (SEQ ID NO: 105);and, c) Eg5 specific siRNA (targeted sequence:5′-AACTGAAGACCTGAAGACAATAA-3′) (SEQ ID NO: 106). SiRNAs were used at 10nM and 1 □g/ml Lipofectamine 2000 final concentration.

The procedure was as follows: The siRNAs were first diluted in OPTIMEM(serum-free transfection media, Invitrogen) at 0.1 uM □M (10-foldconcentrated) and incubated 5-10 min RT. Lipofectamine 2000 was dilutedat 10 □g/ml (10-fold concentrated) for the total number transfectionsand incubated 5-10 minutes at room temperature (RT). Appropriate amountsof diluted 10-fold concentrated Lipofectamine 2000 were mixed 1:1 withdiluted 10-fold concentrated siRNA and incubated at RT for 20-30″(5-fold concentrated transfection solution). 20 □ls of the 5-foldconcentrated transfection solutions were added to the respective samplesand incubated at 37° C. for 96 hours before analysis.

³H-Thymidine incorporation assay: The proliferation assay is a³H-thymidine incorporation method for determining the proliferation ofviable cells by uptake and incorporation of label into DNA.

The procedure was as follows: Cells growing in log phase aretrypsinized, washed, counted and plated in 96-well plates at 1000-4000cells/well in 10% FBS. After 4-8 hrs, the media is replaced. The cellsare incubated for 24-72 hrs, pulsed with ³H-Thy at 1.5 □Ci/ml for 14hrs, harvested onto a filtermat and counted in scintillation cocktail ona Microbeta trilux or other counter.

To address the validation of the 158P3D2 siRNA in reducing theexpression of 158P3D2 protein in cells, Cos-1 cells were transfectedwith pcDNA.3 vector expressing a Myc/His-tagged version of 158P3D2 alone(LF2k) or together with siRNA for either CT1 (bacterial negativecontrol) or 158P3D2 oligo (158P3D2.b). For additional control, a mocktransfection was also included (No DNA). Western blot analysis using anantibody to the Myc tag on 158P3D2 protein showed that the 158P3D2 siRNAsignificantly reduced the expression level of 158P3D2 protein (FIG. 30).These data show that the specific 158P3D2.b siRNA will have utility toprobe the function of 158P3D2 protein in cells.

In order to address the function of 158P3D2 in cells, 158P3D2 wassilenced by transfecting the endogenously expressing 158P3D2 cell lines(SCaBER, a bladder cancer cell line) with the 158P3D2 specific siRNA(158P3D2.b) along with negative siRNA controls (Luc4, targeted sequencenot represented in the human genome) and a positive siRNA control(targeting Eg5) (See FIG. 31). SCaBER cells were shown to express158P3D2 by Northern blot of total cellular RNA. The results indicatedthat when these cells were treated with siRNA specifically targeting the158P3D2 mRNA, the resulting “158P3D2 deficient cells” showed diminishedcell proliferation as measured by this assay (see oligo 158P3D2.btreated cells). This effect is likely caused by an active induction ofapoptosis. The reduced viability is measured by the decreased uptake oflabeled thymidine.

As control, Cos-1 cells, a cell line with no detectable expression of158P3D2 mRNA or protein (by Western blot), was also treated with thepanel of siRNAs (including oligo 158P3D2.b) and no phenotype wasobserved (FIG. 31). This result reflects the fact that the specificprotein knockdown in the SCaBER cells is not a function of generaltoxicity, since the Cos-1 cells did not respond to the 158P3D2.b oligo.The differential response of the two cell lines to the Eg5 control is areflection of differences in levels of cell transfection andresponsiveness of the cell lines to oligo treatment (FIG. 31).

Together, these data indicate that 158P3D2, and variants thereof, playimportant roles in the proliferation of cancer cells and that the lackof 158P3D2 clearly decreases the survival potential of these cells. Itis to be noted that 158P3D2 is constitutively expressed in many tumorcell lines. 158P3D2 serves a role in malignancy; it expression is aprimary indicator of disease, where such disease is often characterizedby high rates of uncontrolled cell proliferation and diminishedapoptosis. Correlating cellular phenotype with gene knockdown followingRNAi treatments is important, and allows one to draw valid conclusionsand rule out toxicity or other non-specific effects of these reagents.To this end, assays to measure the levels of expression of both proteinand mRNA for the target after RNAi treatments are important, includingWestern blotting, FACS staining with antibody, immunoprecipitation,Northern blotting or RT-PCR (Taqman or standard methods). Any phenotypiceffect of the siRNAs in these assays should be correlated with theprotein and/or mRNA knockdown levels in the same cell lines. Knockdownof 158P3D2 is achieved using the 158P3D2.b oligo as measured by Westernblotting and RT-PCR analysis.

Another method to analyze 158P3D2 related cell proliferation isperforming clonogenic assays. In these assays, a defined number of cellsare plated onto the appropriate matrix and the number of colonies formedafter a period of growth following siRNA treatment is counted.

In 158P3D2 cancer target validation, complementing the cellsurvival/proliferation analysis with apoptosis and cell cycle profilingstudies are considered. The biochemical hallmark of the apoptoticprocess is genomic DNA fragmentation, an irreversible event that commitsthe cell to die. A method to observe fragmented DNA in cells is theimmunological detection of histone-complexed DNA fragments by animmunoassay (i.e. cell death detection ELISA) which measures theenrichment of histone-complexed DNA fragments (mono- andoligo-nucleosomes) in the cytoplasm of apoptotic cells. This assay doesnot require pre-labeling of the cells and can detect DNA degradation incells that do not proliferate in vitro (i.e. freshly isolated tumorcells).

The most important effector molecules for triggering apoptotic celldeath are caspases. Caspases are proteases that when activated cleavenumerous substrates at the carboxy-terminal site of an aspartate residuemediating very early stages of apoptosis upon activation. All caspasesare synthesized as pro-enzymes and activation involves cleavage ataspartate residues. In particular, caspase 3 seems to play a centralrole in the initiation of cellular events of apoptosis. Assays fordetermination of caspase 3 activation detect early events of apoptosis.Following RNAi treatments, Western blot detection of active caspase 3presence or proteolytic cleavage of products (i.e. PARP) found inapoptotic cells further support an active induction of apoptosis.Because the cellular mechanisms that result in apoptosis are complex,each has its advantages and limitations. Consideration of othercriteria/endpoints such as cellular morphology, chromatin condensation,membrane blebbing, apoptotic bodies help to further support cell deathas apoptotic. Since not all the gene targets that regulate cell growthare anti-apoptotic, the DNA content of permeabilized cells is measuredto obtain the profile of DNA content or cell cycle profile. Nuclei ofapoptotic cells contain less DNA due to the leaking out to the cytoplasm(sub-G1 population). In addition, the use of DNA stains (i.e., propidiumiodide) also differentiates between the different phases of the cellcycle in the cell population due to the presence of different quantitiesof DNA in G0/G1, S and G2/M. In these studies the subpopulations can bequantified.

For the 158P3D2 gene, RNAi studies facilitate the understanding of thecontribution of the gene product in cancer pathways. Such active RNAimolecules have use in identifying assays to screen for mAbs that areactive anti-tumor therapeutics. Further, siRNA are administered astherapeutics to cancer patients for reducing the malignant growth ofseveral cancer types, including those listed in Table 1. When 158P3D2(and variants) plays a role in cell survival, cell proliferation,tumorigenesis, or apoptosis, it is used as a target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 46 Homology Comparison of 158P3D2 to Known Sequences

The 158P3D2 v.17 protein has 2036 amino acids with a calculatedmolecular weight of 227.6 kDa and a pI of 5.64. 158P3D2 is predicted tobe a predominantly cytoplasmic protein with plasma membrane association(PSORT-II). 158P3D2 contains a single transmembrane region from aminoacids 2000-2022 with high probability that the amino-terminus residesoutside, consistent with the topology of a type I transmembrane protein.Based on the TMpred algorithm of Hofmann and Stoffel which utilizesTMBASE (K. Hofmann, W. Stoffel, TMBASE—A database of membrane spanningprotein segments Biol. Chem. Hoppe-Seyler 374:166, 1993), 158P3D2contains a primary transmembrane region from amino acids 2003-2020(contiguous amino acids with values greater than 0 on the plot have highprobability of being transmembrane regions) with an orientation in whichthe amino terminus resides inside and the carboxyl terminus outside(type II). Another transmembrane algorithm indicated that 158P3D2contains a transmembrane domain from amino acids 2003-2022, with theN-terminus oriented intracellularly consistent with a type II topology.The transmembrane prediction algorithms are accessed through the ExPasymolecular biology server.

By use of the PubMed website of the N.C.B.I., it was found at theprotein level that 158P3D2 v.17 shows 60% homology and 40% identity withhuman otoferlin, a member of the ferlin family of plasma membraneproteins. Further, 158P3D2 v.17 shows 50% homology and 30% identity withdysferlin, another member of the ferlin family, and 80% homology and 75%identity with the murine gene Fer-1-like 4.

The ferlins are a family of transmembrane proteins that have function inmembrane trafficking, including the repair of cell membranes. Mutationof human otoferlin leads to a specific form of nonsyndromic autosomalrecessive deafness (DFNB9) and mutations in dysferlin lead to twosubtypes of muscular dystrophies (reviewed in Bansal, D. and Campbell,K. P., Trends in Cell Biol. 14, 206-213). The major feature of theferlin family includes multiple C2 domains (conserved PKC homologousregion) that function in both Ca²⁺ dependent and Ca²⁺ independentphospholipid binding, as well as protein binding. The mechanism ofaction for dysferlin includes the repair of muscle cell membranedisruptions through dysferlin-containing cell vesicles. Such vesiclesare tethered to the site of membrane tears (where Ca²⁺ concentrationsare increased) via dysferlin molecules that interact with plasmamembrane associated annexin A1 and annexin A2 molecules. The vesiclesprovide the lipid bilayer material to seal the wound.

158P3D2 associates with cell vesicles and the plasma membrane therebyproviding a means for tumor cells to repair membranes during tumorgrowth and metastasis. Such a functional advantage can be exemplified bythe increased stress that tumors experience, including increasedhypoxia, decreased nutrition and increases in free radical formation.These stresses can alter membrane integrity, thereby increasing the needfor robust plasma membrane repair mechanisms. In addition, the C2domains of 158P3D2, and variants thereof, regulate migration of tumorcells expressing this protein. The regulation of the C2 domains occursthrough the phosphorylation of threonine residues (Raftopolou, M., etal., 2004, Science, 303, 1179-81) and modulates the ability of theexpressing cells to migrate. This signal transduction property of the158P3D2 protein expressed in tumor cells enhances their ability tomigrate during metastases, facilitates their homing to distal sites(lymph nodes), and promotes interactions with other cells during theformation of tumor masses. Further, the C2 domains of 158P3D2 play arole in membrane trafficking though interaction with Ca²⁺,phosphatidylserine and phosphatidylcholine (Rizo, J. and Sudhof, T. C.,J. Biol. Chem. 273, 15879-82 (1998). These interactions are crucial forsubsequent membrane metabolism and interaction. Taken together, the158P3D2 protein significantly promotes unregulated growth of cancercells, contributing to their viability and metastatic advantage in vivo.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

1. A composition that comprises: a) a peptide of eight, nine, ten, oreleven contiguous amino acids of a protein of FIG. 2; b) a peptide ofTables VIII-XXI; c) a peptide of Tables XXII to XLV; or, d) a peptide ofTables XLVI to XLIX.
 2. A composition of claim 1, which elicits animmune response.
 3. A protein of claim 2 that is at least 90, 91, 92,93, 94, 95, 96, 97, 98, or 99% homologous or identical to an entireamino acid sequence shown in FIG.
 2. 4. A protein of claim 2, which isbound by an antibody that specifically binds to a protein of FIG.
 2. 5.A composition of claim 2 wherein the composition comprises a cytotoxic Tcell (CTL) polypeptide epitope or an analog thereof, from the amino acidsequence of a protein of FIG.
 2. 6. A composition of claim 5 furtherlimited by a proviso that the epitope is not an entire amino acidsequence of FIG.
 2. 7. A composition of claim 2 further limited by aproviso that the polypeptide is not an entire amino acid sequence of aprotein of FIG.
 2. 8. A composition of claim 2 that comprises anantibody polypeptide epitope from an amino acid sequence of FIG.
 2. 9. Acomposition of claim 8 further limited by a proviso that the epitope isnot an entire amino acid sequence of FIG.
 2. 10. A composition of claim8 wherein the antibody epitope comprises a peptide region of at least 5amino acids of FIG. 2 in any whole number increment up to the end ofsaid peptide, wherein the epitope comprises an amino acid positionselected from: a) an amino acid position having a value greater than 0.5in the Hydrophilicity profile of FIG. 5; b) an amino acid positionhaving a value less than 0.5 in the Hydropathicity profile of FIG. 6; c)an amino acid position having a value greater than 0.5 in the PercentAccessible Residues profile of FIG. 7; d) an amino acid position havinga value greater than 0.5 in the Average Flexibility profile of FIG. 8;e) an amino acid position having a value greater than 0.5 in theBeta-turn profile of FIG. 9; f) a combination of at least two of a)through e); g) a combination of at least three of a) through e); h) acombination of at least four of a) through e); or i) a combination offive of a) through e).
 11. A polynucleotide that encodes a protein ofclaim
 1. 12. A polynucleotide of claim 11 that comprises a nucleic acidmolecule set forth in FIG.
 2. 13. A polynucleotide of claim 12 furtherlimited by a proviso that the encoded protein is not an entire aminoacid sequence of FIG.
 2. 14. A composition comprising a polynucleotidethat is fully complementary to a polynucleotide of claim
 11. 15. An158P3D2 siRNA composition that comprises siRNA (double stranded RNA)that corresponds to the nucleic acid ORF sequence of the 158P3D2 proteinor a subsequence thereof; wherein the subsequence is 19, 20, 21, 22, 23,24, or 25 contiguous RNA nucleotides in length and contains sequencesthat are complementary and non-complementary to at least a portion ofthe mRNA coding sequence.
 16. A polynucleotide of claim 13 that furthercomprises an additional nucleotide sequence that encodes an additionalpeptide of: a) eight, nine, ten, or eleven contiguous amino acids of aprotein of FIG. 2; b) Tables VIII-XXI; c) Tables XXII to XLV; or, d)Tables XLVI to XLIX.
 17. A method of generating a mammalian immuneresponse directed to a protein of FIG. 2, the method comprising:exposing cells of the mammal's immune system to a portion of a) a158P3D2-related protein and/or b) a nucleotide sequence that encodessaid protein, whereby an immune response is generated to said protein.18. A method of generating an immune response of claim 17, said methodcomprising: providing a 158P3D2-related protein that comprises at leastone T cell or at least one B cell epitope; and, contacting the epitopewith a mammalian immune system T cell or B cell respectively, wherebythe T cell or B cell is activated.
 19. A method of claim 18 wherein theimmune system cell is a B cell, whereby the activated B cell generatesantibodies that specifically bind to the 158P3D2-related protein.
 20. Amethod of claim 18 wherein the immune system cell is a T cell that is acytotoxic T cell (CTL), whereby the activated CTL kills an autologouscell that expresses the 158P3D2-related protein.
 21. A method of claim18 wherein the immune system cell is a T cell that is a helper T cell(HTL), whereby the activated HTL secretes cytokines that facilitate thecytotoxic activity of a cytotoxic T cell (CTL) or the antibody-producingactivity of a B cell.
 22. A method for detecting the presence of an mRNAwhich encodes a protein of FIG. 2 in a sample comprising: subjecting thesample to reverse transcription using at least one 158P3D2 cDNA primerwhereby cDNA is produced when mRNA is present in the sample; amplifyingthe cDNA so produced using 158P3D2 polynucleotides as sense andantisense primers; and, detecting the presence of the amplified 158P3D2cDNA; where the presence of amplified 158P3D2 cDNA indicates that anmRNA which encodes a protein of FIG. 2 is present in the sample.
 23. Amethod for detecting, in a sample, the presence of a 158P3D2-relatedprotein or a 158P3D2-related polynucleotide, comprising steps of:contacting the sample with a substance that specifically binds to the158P3D2-related protein or to the 158P3D2-related polynucleotide,respectively, to form a complex; and, determining the presence or amountof the complex in the sample.
 24. A method of claim 23 for detecting thepresence of a 158P3D2-related protein in a sample comprising steps of:contacting the sample with an antibody or fragment thereof either ofwhich specifically binds to the 158P3D2-related protein, and when sobound thereby forms a complex; and, determining the presence or amountof the complex in the sample.
 25. A method of claim 23 for monitoringone or more 158P3D2 gene products in a biological sample, the methodcomprising: determining the status of one or more 158P3D2 gene productsexpressed by cells in a tissue sample from an individual; comparing thestatus so determined to the status of one or more 158P3D2 gene productsin a corresponding normal sample; and, identifying the presence ofaberrant expression status of 158P3D2 in the tissue sample relative tothe normal sample.
 26. The method of claim 25 further comprising a stepof determining if there are one or more elevated gene products of a158P3D2 mRNA or a 158P3D2 protein, whereby the presence of one or moreelevated gene products in the test sample relative to the normal tissuesample indicates the presence or status of a cancer.
 27. A method ofclaim 26 wherein the tissue is selected from a tissue set forth in TableI.
 28. A composition that modulates the status of a cell that expressesa protein of FIG. 2 comprising: a) a substance that modulates the statusof a cell that expresses a protein of FIG. 2, or b) a molecule that iscontrolled by or produced by a protein of FIG.
 2. 29. A 158P3D2 siRNAcomposition according to claim 28 that comprises siRNA (double strandedRNA) that corresponds to the nucleic acid ORF sequence of the 158P3D2protein or a subsequence thereof; wherein the subsequence is 19, 20, 21,22, 23, 24, or 25 contiguous RNA nucleotides in length and containssequences that are complementary and non-complementary to at least aportion of the mRNA coding sequence.
 30. A composition of claim 28,further comprising a physiologically acceptable carrier.
 31. Apharmaceutical composition that comprises the composition of claim 28 ina human unit dose form.
 32. A composition of claim 28 wherein thesubstance comprises an antibody or fragment thereof that specificallybinds to a protein of FIG.
 2. 33. An antibody or fragment thereof ofclaim 32, which is monoclonal.
 34. An antibody of claim 32, which is ahuman antibody, a humanized antibody or a chimeric antibody.
 35. Anon-human transgenic animal that produces an antibody of claim
 32. 36. Ahybridoma that produces an antibody of claim
 33. 37. A composition ofclaim 28 wherein the substance reduces or inhibits the viability, growthor reproduction status of a cell that expresses a protein of FIG.
 2. 38.A composition of claim 28 wherein the substance increases or enhancesthe viability, growth or reproduction status of a cell that expresses aprotein of FIG.
 2. 39. A composition of claim 28 wherein the substanceis selected from the group comprising: a) an antibody or fragmentthereof, either of which immunospecifically binds to a protein of FIG.2; b) a polynucleotide that encodes an antibody or fragment thereof,either of which immunospecifically binds to a protein of FIG. 2; c) aribozyme that cleaves a polynucleotide having a 158P3D2 coding sequence,or a nucleic acid molecule that encodes the ribozyme; and, aphysiologically acceptable carrier; and d) human T cells, wherein said Tcells specifically recognize a 158P3D2 peptide subsequence in thecontext of a particular HLA molecule; e) a protein of FIG. 2, or afragment of a protein of FIG. 2; f) a nucleotide encoding a protein ofFIG. 2, or a nucleotide encoding a fragment of a protein of FIG. 2; g) apeptide of eight, nine, ten, or eleven contiguous amino acids of aprotein of FIG. 2; h) a peptide of Tables VIII-XXI; i) a peptide ofTables XXII to XLV; j) a peptide of Tables XLVI to XLIX; k) an antibodypolypeptide epitope from an amino acid sequence of FIG. 2; l) apolynucleotide that encodes an antibody polypeptide epitope from anamino acid sequence of FIG. 2; or m) an 158P3D2 siRNA composition thatcomprises siRNA (double stranded RNA) that corresponds to the nucleicacid ORF sequence of the 158P3D2 protein or a subsequence thereof;wherein the subsequence is 19, 20, 21, 22, 23, 24, or 25 contiguous RNAnucleotides in length and contains sequences that are complementary andnon-complementary to at least a portion of the mRNA coding sequence. 40.A method of inhibiting viability, growth or reproduction status ofcancer cells that express a protein of FIG. 2, the method comprising:administering to the cells the composition of claim 28, therebyinhibiting the viability, growth or reproduction status of said cells.41. The method of claim 40, wherein the composition comprises anantibody or fragment thereof, either of which specifically bind to a158P3D2-related protein.
 42. The method of claim 40, wherein thecomposition comprises (i) a 158P3D2-related protein or, (ii) apolynucleotide comprising a coding sequence for a 158P3D2-relatedprotein or comprising a polynucleotide complementary to a codingsequence for a 158P3D2-related protein.
 43. The method of claim 40,wherein the composition comprises a ribozyme that cleaves apolynucleotide that encodes a protein of FIG.
 2. 44. The method of claim40, wherein the composition comprises human T cells to said cancercells, wherein said T cells specifically recognize a peptide subsequenceof a protein of FIG. 2 while the subsequence is in the context of theparticular HLA molecule.
 45. The method of claim 40, wherein thecomposition comprises a vector that delivers a nucleotide that encodes asingle chain monoclonal antibody, whereby the encoded single chainantibody is expressed intracellularly within cancer cells that express aprotein of FIG.
 2. 46. A method of delivering an agent to a cell thatexpresses a protein of FIG. 2, said method comprising: providing theagent conjugated to an antibody or fragment thereof of claim 32; and,exposing the cell to the antibody-agent or fragment-agent conjugate. 47.A method of inhibiting viability, growth or reproduction status ofcancer cells that express a protein of FIG. 2, the method comprising:administering to the cells the composition of claim 28, therebyinhibiting the viability, growth or reproduction status of said cells.48. A method of targeting information for preventing or treating acancer of a tissue listed in Table I to a subject in need thereof, whichcomprises: detecting the presence or absence of the expression of apolynucleotide associated with a cancer of a tissue listed in Table I ina sample from a subject, wherein the expression of the polynucleotide isselected from the group consisting of: (a) a nucleotide sequence in FIG.2; (b) a nucleotide sequence which encodes a polypeptide encoded by anucleotide sequence in FIG. 2; (c) a nucleotide sequence which encodes apolypeptide that is 90% or more identical to the amino acid sequenceencoded by a nucleotide sequence in FIG. 2; directing information forpreventing or treating the cancer of a tissue listed in Table I to asubject in need thereof based upon the presence or absence of theexpression of the polynucleotide in the sample.
 49. The method of claim48, wherein the information comprises a description of detectionprocedure or treatment for a cancer of a tissue listed in Table I.
 50. Amethod for identifying a candidate molecule that modulates cellproliferation, which comprises: (a) introducing a test molecule to asystem which comprises a nucleic acid comprising a nucleotide sequenceselected from the group consisting of: (i) the nucleotide sequence ofSEQ ID NO:1; (ii) a nucleotide sequence which encodes a polypeptideconsisting of the amino acid sequence set forth in FIG. 3; (iii) anucleotide sequence which encodes a polypeptide that is 90% or moreidentical to the amino acid sequence set forth in FIG. 3; and (iv) afragment of a nucleotide sequence of (i), (ii), or (iii); or introducinga test molecule to a system which comprises a protein encoded by anucleotide sequence of (i), (ii), (iii), or (iv); and (b) determiningthe presence or absence of an interaction between the test molecule andthe nucleotide sequence or protein, whereby the presence of aninteraction between the test molecule and the nucleotide sequence orprotein identifies the test molecule as a candidate molecule thatmodulates cell proliferation.
 51. The method of claim 50, wherein thesystem is an animal.
 52. The method of claim 50, wherein the system is acell.
 53. The method of claim 50, wherein the test molecule comprises anantibody or antibody fragment that specifically binds the proteinencoded by the nucleotide sequence of (i), (ii), (iii), or (iv).
 54. Amethod for treating a cancer of a tissue listed in Table I in a subject,which comprises administering a candidate molecule identified by themethod of claim 50 to a subject in need thereof, whereby the candidatemolecule treats a cancer of a tissue listed in Table I in the subject.55. A method for identifying a candidate therapeutic for treating acancer of a tissue listed in Table I, which comprises: (a) introducing atest molecule to a system which comprises a nucleic acid comprising anucleotide sequence selected from the group consisting of: (i) thenucleotide sequence of SEQ ID NO:1; (ii) a nucleotide sequence whichencodes a polypeptide consisting of the amino acid sequence set forth inFIG. 3; (iii) a nucleotide sequence which encodes a polypeptide that is90% or more identical to the amino acid sequence set forth in FIG. 3;and (iv) a fragment of a nucleotide sequence of (i), (ii), or (iii); orintroducing a test molecule to a system which comprises a proteinencoded by a nucleotide sequence of (i), (ii), (iii), or (iv); and (b)determining the presence or absence of an interaction between the testmolecule and the nucleotide sequence or protein, whereby the presence ofan interaction between the test molecule and the nucleotide sequence orprotein identifies the test molecule as a candidate therapeutic fortreating a cancer of a tissue listed in Table I.
 56. The method of claim55, wherein the system is an animal.
 57. The method of claim 55, whereinthe system is a cell.
 58. The method of claim 55, wherein the testmolecule comprises an antibody or antibody fragment that specificallybinds the protein encoded by the nucleotide sequence of (i), (ii),(iii), or (iv).